Conjugates for Targeted Cell Surface Editing

ABSTRACT

Provided are conjugates including a targeting moiety that binds to a cell surface molecule of a target cell and a target cell surface-editing enzyme. Also provided are compositions and kits that include the conjugates, as well as methods of using the conjugates. Methods of making conjugates are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication No. 62/357,645 filed Jul. 1, 2016, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with Government support under contracts GM059907and CA108781 awarded by the National Institutes of Health. TheGovernment has certain rights in the invention.

INTRODUCTION

Therapies that enhance the immune response to cancer are provingrevolutionary in the fight against intractable tumors. Immune cellsintegrate signals from activating and inhibitory receptors to determinetheir response to a challenging target—activating signals alert them tothe presence of pathology while inhibitory signals tell the cell that ithas confronted a healthy “self”. Successful tumors evolve mechanisms tothwart immune cell recognition, often by overexpressing ligands forinhibitory receptors. This discovery has led to new therapeuticstrategies aimed at blocking inhibitory immune cell signaling, asembodied in clinically approved T cell checkpoint inhibitors targetingPD-1 and CTLA-4. Ongoing pre-clinical studies have focused on combiningtherapies targeting multiple immunologic pathways. For example,antibodies against PD-1/PD-L1 in combination with those targeting otherT cell checkpoint inhibitors demonstrate improved anti-tumor activity insyngeneic tumor models. A complement to these interventions aretherapies targeting innate immune cells, particularly natural killer(NK) cells, macrophages and dendritic cells.

SUMMARY

Provided are conjugates including a targeting moiety that binds to acell surface molecule of a target cell and a target cell surface-editingenzyme. Also provided are compositions and kits that include theconjugates, as well as methods of using the conjugates. Methods ofmaking conjugates are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates an immune evasion strategy and a methodof reducing such immune evasion according to one embodiment of thepresent disclosure.

FIG. 2 depicts the preparation of antibody-sialidase conjugates andtheir electrophoretic and ESI-MS analysis.

FIG. 3 depicts electrophoretic analysis of sialidases, hydrolysisactivities, and flow cytometry and imaging analysis of their activities

FIG. 4 depicts cell-surface sialylation levels and ligand levels ofvarious receptors with or without sialidase treatment in differentbreast cancer cell lines.

FIG. 5 depicts the cytotoxicity of isolated peripheral blood NK cells inthe absence or presence of sialidase.

FIG. 6 depicts various assays used for the characterization of wild-typeand heat-inactivated V. cholerae sialidase.

FIG. 7 depicts the ESI-MS spectra for Vibrio cholerae sialidase,Salmonella typhimurium sialidase, anti-Her2-IgG and its conjugates.

FIG. 8 depicts the hydrolytic activities of V. cholerae sialidase andanti-Her2-IgG-Sia. Also depicted is hydrolytic activities of S.typhimurium sialidase and anti-Her2-IgG-StSia.

FIG. 9 depicts images showing the level of cell-surface sialic acid invarious cell lines, with or without trastuzumab-sialidase conjugatetreatment. Also shown is flow cytometry data comparing removal ofcell-surface sialic acid by two trastuzumab-sialidase conjugates invarious cell lines.

FIG. 10 depicts images showing Sambucus nigra ligands on various celllines in the absence or presence of anti-Her2-IgG-Sia conjugate.

FIG. 11 depicts the cytotoxicity of isolated peripheral blood NK cellsin the presence of anti-Her2-IgG or anti-Her2-IgG-Sia.

FIG. 12 depicts the activity of trastuzumab and trastuzumab-sialidaseconjugate against HER-2 expressing cancer cells.

FIG. 13 depicts the siglec expression levels and cytotoxicity ofisolated monocyte populations and differentiated macrophages in thepresence of sialidase, trastuzurnab, and trastuzumab-sialidaseconjugates against HER2+ expressing cancer cells.

FIG. 14 depicts the cytotoxicity of isolated γδ T cells in the presenceof sialidase, trastuzunnab, or trastuzumab-sialidase conjugate againstHER2+ expressing cancer cells.

FIG. 15 depicts the cytotoxicity of peripheral blood NK cells withanti-Her2-IgG or a mixture of anti-Her2-IgG and sialidase in the absenceor presence of blocking antibodies as specified.

FIG. 16 depicts flow cytometry data analyzing CD56 and CD3 markers onleukocytes.

FIG. 17 depicts data demonstrating that sialidase treatment potentiatesrituximab-induced complement-dependent cytotoxicity (CDC).

FIG. 18 depicts data showing that Ramos cells have higher levels ofSiglec-9 ligands than Daudi cells.

FIG. 19 depicts data demonstrating that sialidase potentiates rituximabin a complement-dependent manner.

DETAILED DESCRIPTION

Provided are conjugates including a targeting moiety that binds to acell surface molecule of a target cell and a target cell surface-editingenzyme. Also provided are compositions and kits that include theconjugates, as well as methods of using the conjugates. Methods ofmaking conjugates are also provided.

Before the conjugates, compositions and methods of the presentdisclosure are described in greater detail, it is to be understood thatthe conjugates, compositions and methods are not limited to particularembodiments described, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the conjugates, compositions and methodswill be limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the conjugates, compositions andmethods. The upper and lower limits of these smaller ranges mayindependently be included in the smaller ranges and are also encompassedwithin the conjugates, compositions and methods, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the conjugates, compositionsand methods.

Certain ranges are presented herein with numerical values being precededby the term “about.” The term “about” is used herein to provide literalsupport for the exact number that it precedes, as well as a number thatis near to or approximately the number that the term precedes. Indetermining whether a number is near to or approximately a specificallyrecited number, the near or approximating unrecited number may be anumber which, in the context in which it is presented, provides thesubstantial equivalent of the specifically recited number.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the conjugates, compositions and methods belong.Although any conjugates, compositions and methods similar or equivalentto those described herein can also be used in the practice or testing ofthe conjugates, compositions and methods, representative illustrativeconjugates, compositions and methods are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the materials and/or methods in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present conjugates, compositions and methods are notentitled to antedate such publication, as the date of publicationprovided may be different from the actual publication date which mayneed to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the conjugates, compositionsand methods, which are, for clarity, described in the context ofseparate embodiments, may also be provided in combination in a singleembodiment. Conversely, various features of the conjugates, compositionsand methods, which are, for brevity, described in the context of asingle embodiment, may also be provided separately or in any suitablesub-combination. All combinations of the embodiments are specificallyembraced by the present disclosure and are disclosed herein just as ifeach and every combination was individually and explicitly disclosed, tothe extent that such combinations embrace operable processes and/orcompositions. In addition, all sub-combinations listed in theembodiments describing such variables are also specifically embraced bythe present conjugates, compositions and methods and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentmethods. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Conjugates

As summarized above, aspects of the present disclosure includeconjugates. In certain aspects, the conjugates include a targetingmoiety that binds to a cell surface molecule of a target cell, and atarget cell surface-editing enzyme.

Targeting Moieties

According to certain embodiments, a conjugate of the present disclosureincludes a targeting moiety. The targeting moiety may vary and may beselected based, e.g., on the nature of the cell surface molecule on thetarget cell. Non-limiting examples of a targeting moiety that may beemployed include an antibody, a ligand, an aptamer, a nanoparticle, anda small molecule.

In certain aspects, the targeting moiety specifically binds to the cellsurface molecule. As used herein, a targeting moiety that “specificallybinds to the cell surface molecule” or is “specific for the cell surfacemolecule” refers to a targeting moiety that binds the cell surfacemolecule with greater affinity than with other cell surface molecules.According to certain embodiments, the targeting moiety exhibits abinding affinity to the cell surface molecule of a K_(d) of less than orequal to about 10⁻⁵ M, less than or equal to about 10⁻⁶ M, or less thanor equal to about 10⁻⁷ M, or less than or equal to about 10⁻⁸ M, or lessthan or equal to about 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less.Such affinities may be readily determined using conventional techniques,such as by equilibrium dialysis, surface plasmon resonance (SPR)technology (e.g., the BIAcore 2000 instrument, using general proceduresoutlined by the manufacturer), radioimmunoassay, or by another method.

According to certain embodiments, the targeting moiety is an antibody.The terms “antibody” and “immunoglobulin” include antibodies orimmunoglobulins of any isotype (e.g., IgG (e.g., IgG1, IgG2, IgG3, orIgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodiescomposed of a tetramer which in turn is composed of two dimers of aheavy and light chain polypeptide); single chain antibodies; fragmentsof antibodies (e.g., fragments of whole or single chain antibodies)which retain specific binding to the cell surface molecule of the targetcell, including, but not limited to single chain Fv (scFv), Fab,(Fab′)₂, (scFv′)₂, and diabodies; chimeric antibodies; monoclonalantibodies, human antibodies, humanized antibodies (e.g., humanizedwhole antibodies, humanized half antibodies, or humanized antibodyfragments); and fusion proteins comprising an antigen-binding portion ofan antibody and a non-antibody protein. The antibodies may be detectablylabeled, e.g., with an in vivo imaging agent, a radioisotope, an enzymewhich generates a detectable product, a fluorescent protein, and thelike. The antibodies may be further conjugated to other moieties, suchas members of specific binding pairs, e.g., biotin (member ofbiotin-avidin specific binding pair), and the like.

In certain aspects, when the targeting moiety is an antibody, theantibody may be a therapeutic antibody even in the absence of the targetcell surface-editing enzyme, e.g., an antibody having efficacy on itsown in the treatment of cancer (e.g., via antibody-dependent cellularcytotoxicity and/or another mechanism), an immune-related disorder, anendothelial cell-related disorder, or the like. For example, theantibody may be a therapeutic antibody that specifically binds to atumor-associated cell surface molecule or a tumor-specific cell surfacemolecule.

Non-limiting examples of antibodies that specifically bind to atumor-associated cell surface molecule or a tumor-specific cell surfacemolecule which may be employed in a conjugate of the present disclosureinclude Adecatumumab, Ascrinvacumab, Cixutumumab, Conatumumab,Daratumumab, Drozitumab, Duligotumab, Durvalumab, Dusigitumab,Enfortumab, Enoticumab, Figitumumab, Ganitumab, Glembatumumab,Intetumumab, Ipilimumab, Iratumumab, Icrucumab, Lexatumumab,Lucatumumab, Mapatumumab, Narnaturnab, Necitumumab, Nesvacumab,Ofatumumab, Olaratumab, Panitumumab, Patritumab, Pritumumab, Radretumab,Ramucirumab, Rilotumumab, Robatumumab, Seribantumab, Tarextumab,Teprotumumab, Tovetumab, Vantictumab, Vesencumab, Votumumab,Zalutumumab, Flanvotumab, Altumomab, Anatumomab, Arcitumomab,Bectumomab, Blinatumomab, Detumomab, Ibritumomab, Minretumomab,Mitumomab, Moxetumomab, Naptumomab, Nofetumomab, Pemtumomab, Pintumomab,Racotumomab, Satumomab, Solitomab, Taplitumomab, Tenatumomab,Tositumomab, Tremelimumab,

Abagovomab, Igovomab, Oregovomab, Capromab, Edrecolomab, Nacolomab,Amatuximab, Bavituximab, Brentuximab, Cetuximab, Derlotuximab,Dinutuximab, Ensituximab, Futuximab, Girentuximab, Indatuximab,Isatuximab, Margetuximab, Rituximab, Siltuximab, Ublituximab,Ecromeximab, Abituzumab, Alemtuzumab, Bevacizumab, Bivatuzumab,Brontictuzumab, Cantuzumab, Cantuzumab, Citatuzumab, Clivatuzumab,Dacetuzumab, Demcizumab, Dalotuzumab, Denintuzumab, Elotuzumab,Emactuzumab, Emibetuzumab, Enoblituzumab, Etaracizumab, Farletuzumab,Ficlatuzumab, Gemtuzumab, Imgatuzumab, Inotuzumab, Labetuzumab,Lifastuzumab, Lintuzumab, Lorvotuzumab, Lumretuzumab, Matuzumab,Milatuzumab, Nimotuzumab, Obinutuzumab, Ocaratuzumab, Otlertuzumab,Onartuzumab, Oportuzumab, Parsatuzumab, Pertuzumab, Pinatuzumab,Polatuzumab, Sibrotuzumab, Simtuzumab, Tacatuzumab, Tigatuzumab,Trastuzumab, Tucotuzumab, Vandortuzumab, Vanucizumab, Veltuzumab,Vorsetuzumab, Sofituzumab, Catumaxomab, Ertumaxomab, Depatuxizumab,Ontuxizumab, Blontuvetmab, Tamtuvetmab, or an antigen-binding variantthereof. As used herein, “variant” is meant the antibody binds to theparticular antigen (e.g., HER2 for trastuzumab) but has fewer or moreamino acids than the parental antibody, has one or more amino acidsubstitutions relative to the parental antibody, or a combinationthereof.

In certain aspects, the targeting moiety is a therapeutic antibody setforth in Table 1 below approved for treating cancer, or anantigen-binding variant thereof. Also provided in Table 1 is thecorresponding tumor-associated cell surface molecule or tumor-specificcell surface molecule to which the therapeutic antibody specificallybinds, as well as the type of cancer for which the antibody is approvedfor treatment.

TABLE 1 Antibodies approved for treating cancer Cell surface moleculeCancer Types Antibody BCR-ABL CML Imatinib, Dasatinib ALL Nilotinib,Bosutinib Ponatinib CD19 ALL Blinatumomab CD20 NHL, CLL Rituximab B-cellNHL Ofatumumab pre-B ALL ⁹⁰Y-Ibritumomab ¹³¹I-Tositumomab CD30 Hodgkin'slymphoma Brentuximab vedotin CD33 AML Gemtuzumab ozogamicin CD52 CLLAlemtuzumab CTLA-4 Unresectable or metastatic Ipilimumab melanoma EGFRCRC Cetuximab Head and Neck Panitumumab EpCAM Malignant ascitesCatumaxomab HER2 Breast Trastuzumab Pertuzumab PAP Prostate Sipuleucel-TPD-1 Metastatic melanoma NSCLC Nivolumab Pembrolizumab VEGF Breast,Cervical Bevacizumab CRC, NSCLC RCC, Ovarian Glioblastoma VEGF-R2Gastric Ramucirumab NSCLC

Abbreviations for Table 1 are as follows: ALL, acute lymphoblasticleukemia; AML, acute myelogenous leukemia; BCR-ABL, breakpoint clusterregion Abelson tyrosine kinase; CLL, chronic lymphocytic leukemia;CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; CRC, colorectalcancer; EGFR, epidermal growth factor receptor; EpCAM, epithelial celladhesion molecule; HER2, human epidermal growth factor receptor 2; NHL,non-Hodgkin's lymphoma; NSCLC, non-small cell lung cancer; PAP,prostatic acid phosphatase; PD-1, programmed cell death receptor 1; RCC,renal cell carcinoma; VEGF, vascular endothelial growth factor; VEGF-R2,vascular endothelial growth factor receptor 2. In some embodiments, thetargeting moiety is a therapeutic antibody set forth in

Table 2 below or an antigen-binding variant thereof. Also provided inTable 2 is the corresponding tumor-associated cell surface molecule ortumor-specific cell surface molecule to which the therapeutic antibodyspecifically binds, as well as an example cancer type which may betreated using a conjugate that includes the antibody.

TABLE 2 Additional antibodies, cell surface molecules, and cancer typesCell surface molecule Cancer types Antibody A2aR NSCLC PBF-509 AKAP4NSCLC Preclinical Ovarian BAGE Glioblastoma Preclinical Ovarian BORISProstate, Lung Preclinical Esophageal CD22 ALL Epratuzumab MoxetumomabInotuzumab ozogamicin CD73 Advanced solid tumors MEDI9447 CD137 Advancedsolid tumors Urelumab PF-05082566 CEA CRC PANVAC ™ Ad5-[E1-,E2b-]-CEA(6D) CS1 Multiple myeloma Elotuzumab CTLA-4 MalignantTremelimumab mesothelioma EBAG9 Bladder Preclinical EGF NSCLC CIMAvaxEGFR NSCLC Necitumumab GAGE Cervical Preclinical GD2 NeuroblastomaDinutuximab, hu3F8 Retinoblastoma hu14.18-IL-2, 3F8/OKT3BsAb Melanomaother anti-GD2 CAR solid tumors GD2-KLH gp100 Melanoma gp100:209-217(210M) HPV-16 Cervical HPV-16 (E6, E7) SCCHN TG4001, Lm-LLO-E7pNGVL4a-CRT/E7, INO-3112 HSP105 CRC Preclinical Bladder IDH1 GliomaIDH1(R132H) p123-142 Idiotype NSCLC, Breast Racotumomab (NeuGcGM3)Melanoma IDO1 Breast, Melanoma Indoximod INCB024360 NSCLC IDO1 peptidevaccine KIR Lymphoma Lirilumab LAG-3 Breast, Hemato- BMS-986016 logical,Advanced IMP321 solid tumors LY6K Gastric LY6K-177 peptide SCCHN LY6K,CDCA1, IMP3 MAGE-A3 Melanoma recMAGE-A3 NSCLC Zastumotide MAGE-C2Gastric, Melanoma Preclinical Multiple myeloma MAGE-D4 CRC PreclinicalMelan-A Melanoma MART-1 (26-35, 27L) MET NSCLC Onartuzumab TivantinibMUC1 NSCLC, Breast Tecemotide, TG4010 Prostate PANVAC ™ MUC4 PancreaticPreclinical MUC16 Ovarian Abagovomab Oregovomab NY-ESO-1 OvarianNY-ESO-1/ISCOMATRIX ™ Melanoma rV-NY-ESO-1; rF-NY-ESO-1 PD-1 B-celllymphoma Pidilizumab Melanoma, CRC AMP-224, AMP-514 PD-L1 NSCLC, RCCBMS-936559, Atezolizumab Bladder, Breast Durvalumab, Avelumab Melanoma,SCCHN PRAME NSCLC Preclinical PSA Prostate PROSTVAC ®-VF ROR1 CLL,Pancreatic Preclinical Lung, Breast Sialyl-Tn Breast Theratope SPAG-9Prostate, CRC Preclinical NSCLC, Ovarian SSX1 Prostate PreclinicalMultiple myeloma Survivin Melanoma EMD640744 Glioma, Solid tumorsTrivalent peptide vaccine Tripeptide vaccine Telomerase PancreaticTertomotide TIM-3 Melanoma, NHL Preclinical NSCLC VISTA Melanoma,Bladder Preclinical WT1 Ovarian, Uterine, WT1 peptide vaccine AMLMultiple myeloma XAGE-1b Prostate DC-based tumor vaccine 5T4 RCC, CRCTroVax ® Prostate Naptumomab estafenatox

Abbreviations for Table 2 are as follows: A2aR, adenosine A2a receptor;AKAP4, A kinase anchor protein 4; AML, acute myelogenous leukemia; ALL,acute lymphoblastic leukemia; BAGE, B melanoma antigen; BORIS, brotherof the regulator of imprinted sites; CEA, carcinoembryonic antigen; CLL,chronic lymphocytic leukemia; CRC, colorectal cancer; CS1, CD2 subset 1;CTLA-4, cytotoxic T-lymphocyte-associated antigen 4; EBAG9, estrogenreceptor binding site associated antigen 9; EGF, epidermal growthfactor; EGFR, epidermal growth factor receptor; NSCLC, non-small celllung cancer; GAGE, G antigen; GD2, disialoganglioside GD2; gp100,glycoprotein 100; HPV-16, human papillomavirus 16; HSP105, heat-shockprotein 105; IDH1, isocitrate dehydrogenase type 1; IDO1,indoleamine-2,3-dioxygenase 1; KIR, killer cell immunoglobulin-likereceptor; LAG-3, lymphocyte activation gene 3; LY6K, lymphocyte antigen6 complex K; MAGE-A3, melanoma antigen 3; MAGE-C2, melanoma antigen C2;MAGE-D4, melanoma antigen D4; Melan-A/MART-1, melanoma antigenrecognized by T-cells 1; MET, N-methyl-N′-nitroso-guanidine humanosteosarcoma transforming gene; MUC1, mucin 1; MUC4, mucin 4; MUC16,mucin 16; NHL, non-Hodgkin lymphoma; NY-ESO-1, New York esophagealsquamous cell carcinoma 1; PD-1, programmed cell death receptor 1;PD-L1, programmed cell death receptor ligand 1; PRAME, preferentiallyexpressed antigen of melanoma; PSA, prostate specific antigen; RCC,renal cell carcinoma; ROR1, receptor tyrosine kinase orphan receptor 1;SCCHN, squamous cell carcinoma of the head and neck; SPAG-9,sperm-associated antigen 9; SSX1, synovial sarcoma X-chromosomebreakpoint 1; TIM-3, T-cell immunoglobulin domain and mucin domain-3;VISTA, V-domain immunoglobulin-containing suppressor of T-cellactivation; WT1, Wilms' Tumor-1; XAGE-1b, X chromosome antigen 1b.

In some embodiments, the targeting moiety is a therapeutic antibody setforth in Table 3 below or an antigen-binding variant thereof. Alsoprovided in Table 3 is the corresponding tumor-associated cell surfacemolecule or tumor-specific cell surface molecule to which thetherapeutic antibody specifically binds.

TABLE 3 Additional antibodies and corresponding cell surface moleculesAntibody Cell Surface Molecule oregovomab CA125 girentuximab CAIXobinutuzumab CD20 ofatumumab CD20 rituximab CD20 alemtuzumab CD52ipilimumab CTLA-4 tremelimumab CTLA-4 cetuximab EGFR necitumumab EGFRpanitumumab EGFR zalutumumab EGFR edrecolomab EpCAM (17-1A) farletuzumabFR-alpha pertuzumab Her2 trastuzumab Her2 rilotumumab HGF figitumumabIGF-1 ganitumab IGF1R durvalumab IGG1K bavituximab Phosphatidylserineonartuzumab scatter factor receptor kinase bevacizumab VEGF-Aramucirumab VEGFR2

In some embodiments, a conjugate of the present disclosure includes atherapeutic antibody as the targeting moiety selected from trastuzumab,cetuximab, daratumumab, girentuximab, panitumumab, ofatumumab,rituxirnab, and antigen-binding variants thereof. In certain aspects, aconjugate of the present disclosure includes a therapeutic antibody asthe targeting moiety, and the therapeutic antibody is trastuzumab or aHER2-binding variant thereof. The heavy and light chain amino acidsequences of trastuzumab are known and provided in Table 4 below.

TABLE 4 Trastuzunnab heavy and light chain amino acid sequencesTrastuzumab Light Chain DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW(SEQ ID NO: 1) YQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC Trastuzumab Heavy ChainEVOLVESGGGLVQPGGSLRLSCAASGFNIKDIYIH (SEQ ID NO: 2)WVRQAPGKGLEWVARIYPINGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVIVPSSSLGTQTYICNVNHKPS NTKVDKKVEPPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLIVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

When the targeting moiety is an antibody, the target cellsurface-editing enzyme may be conjugated to any suitable region of theantibody. In certain aspects, the targeting moiety is an antibody havinga light chain polypeptide, and the target cell surface-editing enzyme isconjugated to the light chain, e.g., at the C-terminus or an internalregion of the light chain. According to certain embodiments, thetargeting moiety is an antibody having a heavy chain polypeptide, andthe target cell surface-editing enzyme is conjugated to the heavy chain,e.g., at the C-terminus or an internal region of the heavy chain. If theantibody having a heavy chain includes a fragment crystallizable (Fc)region, the target cell surface-editing enzyme may be conjugated to theFc region, e.g., at the C-terminus or an internal region of the Fcregion.

According to certain embodiments, the targeting moiety is a ligand. Asused herein, a “ligand” is a substance that forms a complex with abiomolecule to serve a biological purpose. The ligand may be a substanceselected from a circulating factor, a secreted factor, a cytokine, agrowth factor, a hormone, a peptide, a polypeptide, a small molecule,and a nucleic acid, that forms a complex with the cell surface moleculeon the surface of the target cell. In certain aspects, when thetargeting moiety is a ligand, the ligand is modified in such a way thatcomplex formation with the cell surface molecule occurs, but the normalbiological result of such complex formation does not occur. In certainaspects, the ligand is the ligand of a cell surface receptor present onthe target cell. Cell surface receptors of interest include, but are notlimited to, receptor tyrosine kinases (RTKs), non-receptor tyrosinekinases (non-RTKs), growth factor receptors, etc. When the conjugates ofthe present disclosure include a ligand as the targeting moiety, thetarget cell surface-editing enzyme may be conjugated to any suitableregion of the ligand, e.g., a region of attachment that does notinterfere or substantially interfere with the ability of the ligand tobind (e.g., specifically bind) the target cell surface molecule.

In certain aspects, the targeting moiety is an aptamer. By “aptamer” ismeant a nucleic acid (e.g., an oligonucleotide) that has a specificbinding affinity for the target cell surface molecule. Aptamers exhibitcertain desirable properties for targeted delivery of the target cellsurface-editing enzyme, such as ease of selection and synthesis, highbinding affinity and specificity, low immunogenicity, and versatilesynthetic accessibility. Aptamers that bind to cell surface moleculesare known and include, e.g., TTA1 (a tumor targeting aptamer to theextracellular matrix protein tenascin-C). Aptamers that find use in theconjugates of the present disclosure include those described in Zhu etal. (2015) ChemMedChem 10(1):39-45; Sun et al. (2014) Mol. Ther. NucleicAcids 3:e182; and Zhang et al. (2011) Curr. Med. Chem. 18(27):4185-4194.

According to certain embodiments, the targeting moiety is ananoparticle. As used herein, a “nanoparticle” is a particle having atleast one dimension in the range of from 1 nm to 1000 nm, from 20 nm to750 nm, from 50 nm to 500 nm, including 100 nm to 300 nm, e.g., 120-200nm. The nanoparticle may have any suitable shape, including but notlimited to spherical, spheroid, rod-shaped, disk-shaped, pyramid-shaped,cube-shaped, cylinder-shaped, nanohelical-shaped, nanospring-shaped,nanoring-shaped, arrow-shaped, teardrop-shaped, tetrapod-shaped,prism-shaped, or any other suitable geometric or non-geometric shape. Incertain aspects, the nanoparticle includes on its surface one or more ofthe other targeting moieties described herein, e.g., antibodies,ligands, aptamers, small molecules, etc. Nanoparticles that find use inthe conjugates of the present disclosure include those described in Wanget al. (2010) Pharmacol. Res. 62(2):90-99; Rao et al. (2015) ACS Nano9(6):5725-5740; and Byrne et al. (2008) Adv. Drug Deliv. Rev.60(15):1615-1626.

In certain aspects, the targeting moiety is a small molecule. By “smallmolecule” is meant a compound having a molecular weight of 1000 atomicmass units (amu) or less. In some embodiments, the small molecule is 750amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200amu or less. In certain aspects, the small molecule is not made ofrepeating molecular units such as are present in a polymer. In certainaspects, the target cell surface molecule is a receptor for which theligand is a small molecule, and the small molecule of the conjugate isthe small molecule ligand (or a derivative thereof) of the receptor.Small molecules that find use in targeting a conjugate to a target cellof interest are known. As just one example, folic acid (FA) derivativeshave been shown to effectively target certain types of cancer cells bybinding to the folate receptor, which is overexpressed, e.g., in manyepithelial tumors. See, e.g., Vergote et al. (2015) Ther. Adv. Med.Oncol.

7(4):206-218. In another example, the small molecule sigma-2 has provento be effective in targeting cancer cells. See, e.g., Hashim et al.(2014) Molecular Oncology 8(5):956-967. Sigma-2 is the small moleculeligand for sigma-2 receptors, which are overexpressed in manyproliferating tumor cells including pancreatic cancer cells. In certainaspects, a conjugate of the present disclosure includes a small moleculeas the targeting moiety, in which it has been demonstrated in thecontext of a small molecule drug conjugate (SMDC) that the smallmolecule is effective at targeting a conjugate to a target cell ofinterest by binding to a cell surface molecule on the target cell.

Target Cell Surface Editing Enzymes

As summarized above, the conjugates of the present disclosure include atarget cell surface-editing enzyme. As used herein, a “target cellsurface-editing enzyme” is an enzyme which, upon binding of thetargeting moiety to the corresponding cell surface molecule of thetarget cell, effects a structural change in one or more molecules on thesurface of the target cell. In certain aspects, the structural change isto the cell surface molecule to which the targeting moiety binds. Inother aspects, the structural change is to a molecule on the surface ofthe target cell other than the cell surface molecule to which thetargeting moiety binds.

In certain aspects, the target cell surface-editing enzyme is awild-type enzyme (that is, an enzyme found in nature). In other aspects,the enzyme is not a wild-type enzyme. For example, the target cellsurface-editing enzyme may be a non-natural derivative of a wild-typeenzyme. Such derivatives at least partially retain the enzymaticactivity of the corresponding wild-type enzyme. Enzyme derivatives thatmay be employed include those that have fewer amino acids or more aminoacids than the corresponding wild-type enzyme. Alternatively, oradditionally, the enzyme derivatives may include one or more amino acidsubstitutions or amino acid modifications relative to the correspondingwild-type enzyme.

An example of a structural change effected by a target cellsurface-editing enzyme in a molecule on the surface of the target cellis the cleavage of the molecule. In certain aspects, the moleculecleaved by the target cell surface-editing enzyme is a polymer. Cellsurface polymers which may be cleaved (e.g., degraded) by the targetcell surface-editing enzyme include, but are not limited to,polypeptides, polysaccharides, glycoproteins, and the like. For example,the target cell surface-editing enzyme may be a protease that cleavespolypeptides (or a subgroup of interest thereof) on the surface of thetarget cell. In certain aspects, the polymer cleaved by the target cellsurface-editing enzyme is a polysaccharide (or “glycan”), that is, amolecule containing monosaccharides linked glycosidically. In suchembodiments, the target cell surface-editing enzyme may be, e.g., aglycoside hydrolase (e.g., a sialidase).

According to certain embodiments, the cell surface-editing enzymecleaves (e.g., hydrolyzes) a terminal residue of a molecule (e.g., apolymer) on the surface of the target cell. In certain aspects, theterminal residue is present in a molecule selected from aoligosaccharide, a polysaccharide, a glycoprotein, a glycolipid, and aganglioside. In some embodiments the terminal residue is a terminalsialic acid residue. When the terminal residue is a terminal sialic acidresidue, the cell surface-editing enzyme may be a sialidase (or aderivative thereof as described above), which cleaves the glycosidiclinkages of sialic (neuraminic) acids, releasing terminal sialic acidresidues from oligosaccharides, polysaccharides, glycoproteins,glycolipids, and other substrates.

Sialidases which may be employed in the conjugates of the presentdisclosure include, but are not limited to, prokaryotic sialidases andeukaryotic sialidases. Prokaryotic sialidases that may be employedinclude bacterial sialidases. One example of a bacterial sialidase thatfinds use in the conjugates of the present disclosure is Salmonellatyphimurium sialidase (e.g., UniProtKB-P29768). Another example of abacterial sialidase that finds use in the conjugates of the presentdisclosure is Vibrio cholera sialidase (e.g., UniProtKB-P0C6E9).Eukaryotic sialidases that may be employed include, e.g., mammaliansialidases and non-mammalian eukaryotic sialidases. Mammalian sialidases(or mammalian neuraminidases) of interest include those from primates,e.g., human or non-human neuraminidases. In certain aspects, thesialidase is a human sialidase. According to certain embodiments, thehuman sialidase is selected from human neuraminidase 1 (e.g.,UniProtKB-Q99519), human neuraminidase 2 (e.g., UniProtKB-Q9Y3R4), humanneuraminidase 3 (e.g., UniProtKB-Q9UQ49), and human neuraminidase 4(e.g., UniProtKB-Q8WWR8). It will be understood that the sialidase maybe a derivative of any of the wild-type sialidases above, such astruncated derivatives, derivatives that include more amino acids thanthe corresponding wild-type sialidase, derivatives that include one ormore amino acid substitutions (e.g., one or more conservativesubstitutions, one or more non-conservative substitutions, asubstitution of a natural amino acid with a non-natural amino acid,and/or the like), etc. The derivatives retain at least a portion of theglycoside hydrolase activity of the parental wild-type sialidase.

TABLE 5 Amino acid sequences of example sialidases SalmonellaTVEKSVVFKAEGEHFTDQKGNTIVGSGSGGTTKYFRIPAMCTTS typhimurium sialidaseKGTIVVFADARHNTASDQSFIDTAAARSTDGGKTWNKKIAIYND (SEQ ID NO: 3)RVNSKLSRVMDPTCIVANIQGRETILVMVGKWNNNDKTWGAYRDKAPDTDWDLVLYKSTDDGVTFSKVETNIHDIVTKNGTISAMLGGVGSGLQLNDGKLVFPVQMVRTKNITTVLNTSFIYSTDGITWSLPSGYCEGFGSENNIIEFNASLVNNIRNSGLRRSFETKDFGKTWTEFPPMDKKVDNRNHGVQGSTITIPSGNKLVAAHSSAQNKNNDYTRSDISLYAHNLYSGEVKLIDDFYPKVGNASGAGYSCLSYRKNVDKETLYVVYEANGSIEFQDLSRHLPVIKSYN Vibrio choleraeALFDYNATGDTEFDSPAKQGWMQDNTNNGSGVLTNADGMPA sialidase (SEQ IDWLVQGIGGRAQWTYSLSTNQHAQASSFGWRMTTEMKVLSGG NO: 4)MITNYYANGTQRVLPIISLDSSGNLVVEFEGQTGRTVLATGTAATEYHKFELVFLPGSNPSASFYFDGKLIRDNIQPTASKQNMIVWGNGSSNTDGVAAYRDIKFEIQGDVIFRGPDRIPSIVASSVTPGVVTAFAEKRVGGGDPGALSNTNDIITRTSRDGGITWDTELNLTEQINVSDEFDFSDPRPIYDPSSNTVLVSYARWPTDAAQNGDRIKPWMPNGIFYSVYDVASGNWQAPIDVTDQVKERSFQIAGWGGSELYRRNTSLNSQQDWQSNAKIRIVDGAANQIQVADGSRKYVVTLSIDESGGLVANLNGVSAPIILQSEHAKVHSFHDYELQYSALNHTTTLFVDGQQITTWAGEVSQENNIQFGNADAQIDGRLHVQKIVLTQQGHNLVEFDAFYLAQQTPEVEKDLEKLGWTKIKTGNTMSLYGNASVNPGPGHGITLTRQQNISGSQNGRLIYPAIVLDRFFLNVMSIYSDDGGSNWQTGSTLPIPFRWKSSSILETLEPSEADMVELQNGDLLLTARLDFNQIVNGVNYSPRQQFLSKDGGITWSLLEANNANVFSNISTGTVDASITRFEQSDGSHFLLFTNPQGNPAGTNGRQNLGLWFSFDEGVTWKGPIQLVNGASAYSDIYQLDSENAIVIVETDNSNMRIL RMPITLLKQKLTLSQN

According to certain embodiments, the target cell surface-editing enzymeedits (e.g., cleaves all or a portion of) a ligand on the surface of thetarget cell. In some embodiments, the ligand is a ligand of an immunereceptor. Immune receptor ligands of interest include, but are notlimited to, ligands of inhibitory immune receptors. In certain aspects,the target cell surface-editing enzyme cleaves a ligand of an inhibitoryimmune receptor, where the inhibitory immune receptor is present on acell selected from a natural killer (NK) cell, a macrophage, a monocyte,a neutrophil, a dendritic cell, a T cell, a B cell, a mast cell, abasophil, and an eosinophil. By way of example, the ligand on thesurface of the target cell edited by the target cell surface-editingenzyme may be a ligand for a sialic acid-binding Ig-like lectin (Siglec)receptor, e.g., Siglec 7, Siglec 9, and/or the like. According tocertain embodiments, such a ligand is a sialoglycan.

In certain aspects, a structural change in a molecule on the surface ofthe target cell effected by the target cell surface-editing enzyme isthe oxidation of the molecule.

In some embodiments, the structural change in a molecule on the surfaceof the target cell effected by the target cell surface-editing enzyme isthe reduction of the molecule.

In certain aspects, the target cell surface-editing enzyme effects astructural change in a molecule on the surface of the target cell byadding a moiety to the molecule. For example, the target cellsurface-editing enzyme may be a transferase that transfers a functionalgroup to the molecule from a donor molecule. In some embodiments, thetarget cell surface-editing enzyme is a kinase that adds a phosphategroup to the molecule on the surface of the target cell.

According to some embodiments, the target cell surface-editing enzymeeffects a structural change in a molecule on the surface of the targetcell by removing a moiety from the molecule. For example, the targetcell surface-editing enzyme may be a transferase that transfers afunctional group from the molecule to an acceptor molecule. In someembodiments, the target cell surface-editing enzyme is a phosphatasethat removes a phosphate group from the molecule on the surface of thetarget cell.

Target Cells

The targeting moiety and target cell surface-editing enzyme may beselected based on the cell to be targeted. According to certainembodiments, the target cell is selected from a cancer cell, an immunecell, an endothelial cell, and an epithelial cell. Target cells ofinterest include, but are not limited to, cells that are relevant to aparticular disease or condition. For example, the target cell may be anormal functioning cell (e.g., a normal functioning immune cell, etc.),and the cell surface editing enzyme modulates the function of the cellin a manner that is therapeutic to an individual in need thereof, e.g.,boosts a function of the cell that is beneficial in treating a diseasein an individual.

In other aspects, the target cell is not a normal cell. Non-normaltarget cells of interest include, but are not limited to, cancer cells.By “cancer cell” is meant a cell exhibiting a neoplastic cellularphenotype, which may be characterized by one or more of, for example,abnormal cell growth, abnormal cellular proliferation, loss of densitydependent growth inhibition, anchorage-independent growth potential,ability to promote tumor growth and/or development in animmunocompromised non-human animal model, and/or any appropriateindicator of cellular transformation. “Cancer cell” may be usedinterchangeably herein with “tumor cell”, “malignant cell” or “cancerouscell”, and encompasses cancer cells of a solid tumor, a semi-solidtumor, a primary tumor, a metastatic tumor, and the like. In certainaspects, the cancer cell is a carcinoma cell. According to certainembodiments, the cancer cell is selected from a breast cancer cell, anovarian cancer cell, a gastric cancer cell, a colon cancer cell, and acancer cell of any of the cancer types set forth in Tables 1 and 2above.

In certain aspects, when the target cell is a cancer cell, the moleculeon the surface of the cancer cell to which the targeting moiety binds isa tumor-associated cell surface molecule or a tumor-specific cellsurface molecule. By “tumor-associated cell surface molecule” is meant acell surface molecule expressed on malignant cells with limitedexpression on cells of normal tissues, a cell surface molecule expressedat much higher density on malignant versus normal cells, or a cellsurface molecule that is developmentally expressed.

When the target cell is a cancer cell, the cancer cell may express atumor-associated cell surface molecule or tumor-specific cell surfacemolecule to which the targeting moiety binds. In certain aspects, such acell surface molecule is selected from HER2, CD19, CD22, CD30, CD33,CD56, CD66/CEACAM5, CD70, CD74, CD79b, CD138, Nectin-4, Mesothelin,Transmembrane glycoprotein NMB (GPNMB), Prostate-Specific MembraneAntigen (PSMA), SLC44A4, CA6, CA-IX, an integrin, C-X-C chemokinereceptor type 4 (CXCR4), cytotoxic T-lymphocyte-associated protein 4(CTLA-4), neuropilin-1 (NRP1), matriptase, any cell surface molecule setforth in Tables 1, 2, and 3 above, and any other tumor-associated ortumor-specific cell surface molecules of interest.

Methods of Making Conjugates

Methods of making the conjugates of the present disclosure are alsoprovided.

In cases where one wishes to produce the targeting moiety and/or thetarget cell surface-editing enzyme (e.g., because a particular targetingmoiety and/or target cell surface-editing enzyme is not commerciallyavailable), the methods may include producing one or both of thetargeting moiety and target cell surface-editing enzyme. When acomponent of the desired conjugate (that is, the targeting moiety ortarget cell surface-editing enzyme) is a peptide or polypeptide,recombinant methods can be used to produce the component. For example, aDNA encoding a component of the desired conjugate can be inserted intoan expression vector. The DNA encoding the component may be operablylinked to one or more control sequences in the expression vector thatensure the expression of the component. Expression control sequencesinclude, but are not limited to, promoters (e.g., naturally-associatedor heterologous promoters), signal sequences, enhancer elements, andtranscription termination sequences. The expression control sequencescan be promoter systems in vectors capable of transforming ortransfecting prokaryotic or eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the component.

When a component of the desired conjugate (that is, the targeting moietyor target cell surface-editing enzyme) is a peptide or polypeptide, thecomponent may be produced using a chemical peptide synthesis technique.Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid-phase or solid-phase. Solid phase polypeptide synthesis(SPPS), in which the C-terminal amino acid of the sequence is attachedto an insoluble support followed by sequential addition of the remainingamino acids in the sequence, is an example of a suitable method for thechemical synthesis of a component of the desired conjugate. Variousforms of SPPS, such as Fmoc and Boc, are available for synthesizing thecomponent. Briefly, small insoluble, porous beads may be treated withfunctional units on which peptide chains are built. After repeatedcycling of coupling/deprotection, the free N-terminal amine of asolid-phase attached is coupled to a single N-protected amino acid unit.This unit is then deprotected, revealing a new N-terminal amine to whicha further amino acid may be attached. The peptide remains immobilized onthe solid-phase and undergoes a filtration process before being cleavedoff.

Once synthesized (either chemically or recombinantly), the component canbe purified according to standard procedures of the art, includingammonium sulfate precipitation, affinity columns, column chromatography,high performance liquid chromatography (HPLC) purification, gelelectrophoresis, and the like.

Once the targeting moiety and target cell surface-editing enzyme areobtained, a variety of conjugation strategies are available, and aparticular method may be selected based on the nature/type of targetingmoiety and target cell surface-editing enzyme in the desired conjugate(e.g., based on available, or provided, reactive functional groups inthe targeting moiety and target cell surface-editing enzyme).Bioconjugation strategies that find use in stably associating atargeting moiety and a target cell surface-editing enzyme to produce aconjugate of the present disclosure include those described inHermanson, “Bioconjugate Techniques,” Academic Press, 2nd edition, Apr.1, 2008, Haugland, 1995, Methods Mol. Biol. 45:205-21; Brinkley, 1992,Bioconjugate Chemistry 3:2, and elsewhere.

According to certain embodiments, the targeting moiety and target cellsurface-editing enzyme are directly conjugated to each other—that is,the components of the conjugate are conjugated to each other without theuse of a linker. In other aspects, the targeting moiety and target cellsurface-editing enzyme are conjugated to each other via a linker. Anysuitable linker(s) may be employed. Linkers that find use in theconjugates of the present disclosure include ester linkers, amidelinkers, maleimide or maleimide-based linkers; valine-citrullinelinkers; hydrazone linkers; N-succinimidyl-4-(2-pyridyldithio)butyrate(SPDB) linkers;Succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)linkers; vinylsulfone-based linkers; linkers that include polyethyleneglycol (PEG), such as, but not limited to tetraethylene glycol; linkersthat include propanoic acid; linkers that include caproleic acid, andlinkers including any combination thereof. In certain aspects, thelinker includes polyethylene glycol (PEG). In some embodiments, thelinker is a peptide linker. The peptide linker may be flexible or rigid.Peptide linkers of interest include, but are not limited to, thosedescribed in Chen et al. (2013) Adv. Drug Deliv. Rev. 65(10):1357-1369.In certain aspects, when the linker is a peptide linker, the conjugateis a fusion protein. When the conjugate is a fusion protein, the presentdisclosure further provides nucleic acids that encode such fusionproteins, expression vectors that include such nucleic acids operablylinked to a promoter, and host cells (e.g., mammalian host cells) thatinclude such fusion proteins, nucleic acids, and/or expression vectors.In certain aspects, the linker is serum-stable. Serum-stable linkers areknown and include, e.g., linkers that include PEG, sulfone linkers(e.g., phenyloxadiazole sulfone linkers (see Patterson et al. (2014)Bioconj. Chem. 25(8):1402-7)), and the like.

Numerous strategies are available for linking the targeting moiety andtarget cell surface-editing enzyme via a linker. For example, onecomponent of the conjugate may be derivatized by covalently attaching alinker to the component, where the linker has a functional group capableof reacting with a “chemical handle” on that component, and where thelinker has a second functional group capable of reacting with a“chemical handle” on the other component. The functional groups on thelinker may vary and may be selected based on compatibility with thechemical handles on the components of the desired conjugate. Theconjugate components may already include a functional group useful forreacting with a functional group of the linker, or such a functionalgroup may be provided to one or both components of the desiredconjugate. Functional groups that may be used to bind components of theconjugates to a linker include, but are not limited to, active esters,isocyanates, imidoesters, hydrazides, amino groups, aldehydes, ketones,photoreactive groups, maleimide groups, alpha-halo-acetyl groups,epoxides, azirdines, and the like. Reagents such as iodoacetamides,maleimides, benzylic halides and bromomethylketones react byS-alkylation of thiols to generate stable thioether products. Forexample, at pH 6.5-7.5, maleimide groups react with sulfhydryl groups toform stable thioether bonds. Arylating reagents such as NBD halidesreact with thiols or amines by a similar substitution of the aromatichalide by the nucleophile. Because the thiolate anion is a betternucleophile than the neutral thiol, cysteine is more reactive above itspK_(a) (˜8.3, depending on protein structural context). Thiols alsoreact with certain amine-reactive reagents, including isothiocyanatesand succinimidyl esters. The TS-Link series of reagents are availablefor reversible thiol modification.

With respect to amine reactive groups, primary amines exist at theN-terminus of polypeptide chains and in the side-chain of lysine (Lys,K) amino acid residues. Among the available functional groups in typicalbiological or protein samples, primary amines are especiallynucleophilic, making them ready targets for conjugation with severalreactive groups. For example, NHS esters are reactive groups formed bycarbodiimide-activation of carboxylate molecules. NHS ester-activatedcrosslinkers and labeling compounds react with primary amines inphysiologic to slightly alkaline conditions (pH 7.2 to 9) to yieldstable amide bonds. The reaction releases N-hydroxysuccinimide (NHS).Also by way of example, imidoester crosslinkers react with primaryamines to form amidine bonds. Imidoester crosslinkers react rapidly withamines at alkaline pH but have short half-lives. As the pH becomes morealkaline, the half-life and reactivity with amines increases. As such,crosslinking is more efficient when performed at pH 10 than at pH 8.Reaction conditions below pH 10 may result in side reactions, althoughamidine formation is favored between pH 8-10.

Numerous other synthetic chemical groups will form chemical bonds withprimary amines, including but not limited to, isothiocyanates,isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals,epoxides, oxiranes, carbonates, aryl halides, carbodiim ides,anhydrides, and fluorophenyl esters. Such groups conjugate to amines byeither acylation or alkylation.

According to one embodiment, the chemical handle on the targetingmoiety, target cell surface-editing enzyme, or both, is provided byincorporation of an unnatural amino acid having the chemical handle intothe component. The unnatural amino acid may be incorporated via chemicalsynthesis or recombinant approaches, e.g., using a suitable orthogonalamino acyl tRNA synthetase-tRNA pair for incorporation of the unnaturalamino acid during translation in a host cell. The functional group of anunnatural amino acid present in the component may be an azide, alkyne,alkene, amino-oxy, hydrazine, aldehyde, nitrone, nitrile oxide,cyclopropene, norbornene, iso-cyanide, aryl halide, boronic acid, orother suitable functional group, and the functional group on the linkeris selected to react with the functional group of the unnatural aminoacid (or vice versa).

In other aspects, the chemical handle on the targeting moiety, targetcell surface-editing enzyme, or both, is provided using an approach thatdoes not involve an unnatural amino acid. For example, a componentcontaining no unnatural amino acid(s) could be conjugated to a linker byutilizing, e.g., nucleophilic functional groups of the component (suchas the N-terminal amine or the primary amine of lysine, or any othernucleophilic amino acid residue) as a nucleophile in a substitutionreaction with a linker construct bearing a reactive leaving group orother electrophilic group.

In certain aspects, the target cell surface-editing enzyme issite-specifically conjugated to the targeting moiety, the targetingmoiety is site-specifically conjugated to the target cellsurface-editing enzyme, or both. In some embodiments, site-specificconjugation is achieved by incorporating an unnatural amino acid havingthe reactive functional group at a predetermined location in thetargeting moiety and/or target cell surface-editing enzyme.

Details for site-specific incorporation of unnatural amino acids intoproteins can be found, e.g., in Young & Schultz (2010) J. Biol. Chem.285:11039-11044.

In certain aspects, the targeting moiety has a C-terminal aldehyde tag,and site-specific conjugation is achieved by reacting the C-terminalaldehyde with aminooxy-tetraethyleneglycol-azide (aminooxy-TEG-N₃),followed by reacting with a bicyclononyne-N-hydroxysuccinimde ester(BCN-NHS)-labeled target cell surface-editing enzyme. This exampleembodiment is described in more detail in the Experimental sectionbelow.

In certain aspects, the targeting moiety has a C-terminal aldehyde tag,and site specific conjugation is achieved by reacting the C-terminalaldehyde with aminooxy-tetraethyleneglycol-azide (aminooxy-TEG-N₃). Atarget cell surface editing enzyme has an aldehyde tag sequence(SLCTPSRGS), and site-specific conjugation is achieved by reactingaldehyde tag cysteine withDibenzocyclooctyne-tetrapolyethyleneglycol-maleim ide(DBCO-PEG4-maleimide) followed by reaction with the TEG-N₃-labeledtargeting moiety. This example embodiment is described in more detail inthe Experimental section below.

Accordingly, aspects of the present disclosure include methods thatinclude conjugating a target cell surface-editing enzyme to a targetingmoiety that binds to a cell surface molecule on the surface of a targetcell. One or more (e.g., two or more, three or more, four or more, etc.)target cell surface-editing enzymes may be conjugated to the targetingmoiety. The targeting moiety and target cell surface-editing enzyme maybe any of the targeting moieties and target cell surface-editing enzymesdescribed herein. As just one example, in some embodiments, the targetcell surface-editing enzyme is a sialidase (e.g., any of the sialidasesdescribed herein) and the targeting moiety is an antibody (e.g., any ofthe antibodies described herein, including, by way of example, ananti-HER2 antibody (e.g., trastuzamab), cetuximab, daratumumab,girentuximab, panitumumab, ofatumumab, rituximab, etc.). As noted above,the conjugation may be site-specific (e.g., via a functional group of anon-natural amino acid at a predetermined position) with respect to thetargeting moiety, the target cell surface-editing enzyme, or both.

Compositions

Also provided are compositions that include a conjugate of the presentdisclosure. The compositions may include any of the conjugates describedherein (e.g., a conjugate having any of the targeting moieties andtarget cell surface-editing enzymes described herein). In certainaspects, the compositions include a conjugate of the present disclosurepresent in a liquid medium. The liquid medium may be an aqueous liquidmedium, such as water, a buffered solution, or the like. One or moreadditives such as a salt (e.g., NaCl, MgCl₂, KCl, MgSO₄), a bufferingagent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonicacid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS),N-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), asolubilizing agent, a detergent (e.g., a non-ionic detergent such asTween-20, etc.), a ribonuclease inhibitor, glycerol, a chelating agent,and the like may be present in such compositions.

Pharmaceutical corn positions are also provided. The pharmaceutical cornpositions include any of the conjugates of the present disclosure, and apharmaceutically acceptable carrier. The pharmaceutical compositionsgenerally include a therapeutically effective amount of the conjugate.By “therapeutically effective amount” is meant a dosage sufficient toproduce a desired result, e.g., an amount sufficient to effectbeneficial or desired therapeutic (including preventative) results, suchas a reduction in a symptom of a disease or disorder associated with thetarget cell or a population thereof, as compared to a control. Aneffective amount can be administered in one or more administrations.

A conjugate of the present disclosure can be incorporated into a varietyof formulations for therapeutic administration. More particularly, theconjugate can be formulated into pharmaceutical corn positions bycombination with appropriate, pharmaceutically acceptable excipients ordiluents, and may be formulated into preparations in solid, semi-solid,liquid or gaseous forms, such as tablets, capsules, powders, granules,ointments, solutions, injections, inhalants and aerosols.

Formulations of the conjugates of the present disclosure suitable foradministration to a patient (e.g., suitable for human administration)are generally sterile and may further be free of detectable pyrogens orother contaminants contraindicated for administration to a patientaccording to a selected route of administration.

In pharmaceutical dosage forms, the conjugates can be administered inthe form of their pharmaceutically acceptable salts, or they may also beused alone or in appropriate association, as well as in combination,with other pharmaceutically active compounds, e.g., an anti-cancer agent(including but not limited to small molecule anti-cancer agents), animmune checkpoint inhibitor, and any combination thereof. The followingmethods and carriers/excipients are merely examples and are in no waylimiting.

For oral preparations, the conjugate can be used alone or in combinationwith appropriate additives to make tablets, powders, granules orcapsules, for example, with conventional additives, such as lactose,mannitol, corn starch or potato starch; with binders, such ascrystalline cellulose, cellulose derivatives, acacia, corn starch orgelatins; with disintegrators, such as corn starch, potato starch orsodium carboxymethylcellulose; with lubricants, such as talc ormagnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

The conjugate can be formulated for parenteral (e.g., intravenous,intra-arterial, intraosseous, intramuscular, intracerebral,intracerebroventricular, intrathecal, subcutaneous, etc.)administration. In certain aspects, the conjugate is formulated forinjection by dissolving, suspending or emulsifying the conjugate in anaqueous or non-aqueous solvent, such as vegetable or other similar oils,synthetic aliphatic acid glycerides, esters of higher aliphatic acids orpropylene glycol; and if desired, with conventional additives such assolubilizers, isotonic agents, suspending agents, emulsifying agents,stabilizers and preservatives.

Pharmaceutical compositions that include the conjugate may be preparedby mixing the conjugate having the desired degree of purity withoptional physiologically acceptable carriers, excipients, stabilizers,surfactants, buffers and/or tonicity agents. Acceptable carriers,excipients and/or stabilizers are nontoxic to recipients at the dosagesand concentrations employed, and include buffers such as phosphate,citrate, and other organic acids; antioxidants including ascorbic acid,glutathione, cysteine, methionine and citric acid; preservatives (suchas ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methylor propyl parabens, benzalkonium chloride, or combinations thereof);amino acids such as arginine, glycine, ornithine, lysine, histidine,glutamic acid, aspartic acid, isoleucine, leucine, alanine,phenylalanine, tyrosine, tryptophan, methionine, serine, proline andcombinations thereof; monosaccharides, disaccharides and othercarbohydrates; low molecular weight (less than about 10 residues)polypeptides; proteins, such as gelatin or serum albumin;

chelating agents such as EDTA; sugars such as trehalose, sucrose,lactose, glucose, mannose, maltose, galactose, fructose, sorbose,raffinose, glucosamine, N-methylglucosamine, galactosamine, andneuraminic acid; and/or non-ionic surfactants such as Tween, BrijPluronics, Triton-X, or polyethylene glycol (PEG).

The pharmaceutical composition may be in a liquid form, a lyophilizedform or a liquid form reconstituted from a lyophilized form, wherein thelyophilized preparation is to be reconstituted with a sterile solutionprior to administration. The standard procedure for reconstituting alyophilized composition is to add back a volume of pure water (typicallyequivalent to the volume removed during lyophilization); howeversolutions comprising antibacterial agents may be used for the productionof pharmaceutical compositions for parenteral administration.

An aqueous formulation of the conjugate may be prepared in a pH-bufferedsolution, e.g., at pH ranging from about 4.0 to about 7.0, or from about5.0 to about 6.0, or alternatively about 5.5. Examples of buffers thatare suitable for a pH within this range include phosphate-, histidine-,citrate-, succinate-, acetate-buffers and other organic acid buffers.The buffer concentration can be from about 1 mM to about 100 mM, or fromabout 5 mM to about 50 mM, depending, e.g., on the buffer and thedesired tonicity of the formulation.

A tonicity agent may be included in the formulation to modulate thetonicity of the formulation. Example tonicity agents include sodiumchloride, potassium chloride, glycerin and any component from the groupof amino acids, sugars as well as combinations thereof. In someembodiments, the aqueous formulation is isotonic, although hypertonic orhypotonic solutions may be suitable. The term “isotonic” denotes asolution having the same tonicity as some other solution with which itis compared, such as physiological salt solution or serum. Tonicityagents may be used in an amount of about 5 mM to about 350 mM, e.g., inan amount of 100 mM to 350 mM.

A surfactant may also be added to the formulation to reduce aggregationand/or minimize the formation of particulates in the formulation and/orreduce adsorption. Example surfactants include polyoxyethylensorbitanfatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij),alkylphenylpolyoxyethylene ethers (Triton-X),polyoxyethylene-polyoxypropylene copolymer (Poloxamer, Pluronic), andsodium dodecyl sulfate (SDS). Examples of suitablepolyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (soldunder the trademark Tween 2™) and polysorbate 80 (sold under thetrademark Tween 80™). Examples of suitable polyethylene-polypropylenecopolymers are those sold under the names Pluronic® F68 or Poloxamer188™. Examples of suitable Polyoxyethylene alkyl ethers are those soldunder the trademark Brij™. Example concentrations of surfactant mayrange from about 0.001% to about 1% w/v.

A lyoprotectant may also be added in order to protect the conjugateagainst destabilizing conditions during a lyophilization process. Forexample, known lyoprotectants include sugars (including glucose andsucrose); polyols (including mannitol, sorbitol and glycerol); and aminoacids (including alanine, glycine and glutamic acid). Lyoprotectants canbe included in an amount of about 10 mM to 500 nM.

In some embodiments, the pharmaceutical composition includes a conjugateof the present disclosure, and one or more of the above-identifiedagents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent)and is essentially free of one or more preservatives, such as ethanol,benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propylparabens, benzalkonium chloride, and combinations thereof. In otherembodiments, a preservative is included in the formulation, e.g., atconcentrations ranging from about 0.001 to about 2% (w/v).

Methods

As summarized above, methods of using the conjugates of the presentdisclosure are also provided. In certain aspects, the methods of thepresent disclosure include administering to an individual in needthereof a therapeutically effective amount of any of the conjugates ofthe present disclosure, or any of the pharmaceutical compositions of thepresent disclosure.

In certain aspects, the administering modulates an immune pathway in theindividual. For example, the administering may modulate an immunepathway selected from an inhibitory immune receptor pathway, acomplement pathway, a paired immunoglobulin-like type 2 receptor (PILR)pathway, and a natural-killer group 2, member D protein (NKG2D) pathway.In certain aspects, the target cell includes a ligand on its surface,and the administering results in editing of the ligand by the targetcell surface-editing enzyme. The ligand may be edited in any mannerdescribed elsewhere herein. According to certain embodiments, theediting of the ligand comprises cleavage of all or a portion of theligand. As just one example, the ligand may be a sialoglycan, the targetcell surface-editing enzyme may be a sialidase, and the editing mayinclude cleavage of a terminal sialic acid residue of the sialoglycan.The sialidase of the conjugate may be a bacterial sialidase, a mammalianneuraminidase, or the like. When the sialidase is a mammalianneuraminidase, the mammalian neuraminidase may be a human neuraminidase,e.g., a human neuraminidase selected from human neuraminidase 1, humanneuraminidase 2, human neuraminidase 3, and human neuraminidase 4.

When the administering results in editing of a ligand on the target cellby the target cell surface-editing enzyme, the ligand may be a ligand ofan inhibitory immune receptor. In certain aspects, the ligand is aligand of an inhibitory immune receptor present on an immune cellselected from the group consisting of: a natural killer (NK) cell, amacrophage, a monocyte, a neutrophil, a dendritic cell, a T cell, a Bcell, a mast cell, a basophil, and an eosinophil. In some embodiments,the inhibitory immune receptor is a sialic acid-binding Ig-like lectin(Siglec) receptor.

In certain aspects, the methods of the present disclosure includeadministering the conjugate or pharmaceutical composition to anindividual having cancer, e.g., to treat the cancer. Cancers which maybe treated according to the methods of the present disclosure include,but are not limited to, any of the cancers set forth in Tables 1 and 2above. The conjugate may include a targeting moiety (e.g., a therapeuticantibody, such as any of the antibodies set forth in Tables 1, 2, and 3above) that binds to a tumor-associated cell surface molecule ortumor-specific cell surface molecule on the surface of a cancer cell ofthe individual. In some embodiments, the cancer cell is a carcinomacell. According to certain embodiments, the cancer cell is selected froma breast cancer cell, an ovarian cancer cell, a gastric cancer cell, acolon cancer cell, and a cancer cell of any of the cancer types setforth in Tables 1 and 2 above. In certain aspects, the cell surfacemolecule is human epidermal growth factor receptor 2 (HER2). When thecell surface molecule is HER2, the targeting may be, e.g., an anti-HER2antibody (e.g., trastuzamab or another suitable anti-HER2 antibody).

In some embodiments, the administering includes administering aconjugate or pharmaceutical composition of the present disclosure, andthe conjugate includes a targeting moiety that is an antibody. Incertain aspects, the individual in need thereof has cancer, thetargeting moiety of the conjugate is an antibody set forth in Table 1,and the methods are for treating (e.g., by enhanced antibody-dependentcellular cytotoxicity (ADCC)) the same or different type of cancercorresponding to the antibody as set forth in Table 1.

In certain aspects, the administering includes administering a conjugateor pharmaceutical composition of the present disclosure, and theconjugate includes a targeting moiety that is an antibody. In someembodiments, the individual in need thereof has cancer, the targetingmoiety of the conjugate is an antibody set forth in Table 2, and themethods are for treating (e.g., by enhanced antibody-dependent cellularcytotoxicity (ADCC)) the same or different type of cancer correspondingto the antibody as set forth in Table 2.

In some embodiments, the administering includes administering aconjugate or pharmaceutical composition of the present disclosure, andthe conjugate includes a targeting moiety that is an antibody. Incertain aspects, the individual in need thereof has cancer, thetargeting moiety of the conjugate is an antibody set forth in Table 3,and the methods are for treating the cancer (e.g., by enhancedantibody-dependent cellular cytotoxicity (ADCC)).

In certain aspects, the administering includes administering a conjugateor pharmaceutical composition of the present disclosure, and theconjugate includes a targeting moiety that is an antibody selected fromtrastuzamab, cetuximab, daratumumab, girentuximab, panitumumab,ofatumumab, and rituximab.

The conjugates of the present disclosure are administered to theindividual using any available method and route suitable for drugdelivery, including in vivo and ex vivo methods, as well as systemic andlocalized routes of administration. Conventional and pharmaceuticallyacceptable routes of administration include intranasal, intramuscular,intra-tracheal, subcutaneous, intradermal, topical application, ocular,intravenous, intra-arterial, nasal, oral, and other enteral andparenteral routes of administration. Routes of administration may becombined, if desired, or adjusted depending upon the conjugate and/orthe desired effect. The conjugate may be administered in a single doseor in multiple doses. In some embodiments, the conjugate is administeredorally. In some embodiments, the conjugate is administered via aninhalational route. In some embodiments, the conjugate is administeredintranasally. In some embodiments, the conjugate is administeredlocally. In some embodiments, the conjugate is administered ocularly. Insome embodiments, the conjugate is administered intracranially. In someembodiments, the conjugate is administered intravenously. In someembodiments, the conjugate is administered by injection, e.g., forsystemic delivery (e.g., intravenous infusion) or to a local site.

A variety of individuals are treatable according to the subject methods.Generally such individuals are “mammals” or “mammalian,” where theseterms are used broadly to describe organisms which are within the classmammalia, including the orders carnivore (e.g., dogs and cats), rodentia(e.g., mice, guinea pigs, and rats), and primates (e.g., humans,chimpanzees, and monkeys). In some embodiments, the individual is ahuman.

By “treat” or “treatment” is meant at least an amelioration of thesymptoms associated with the pathological condition afflicting theindividual, where amelioration is used in a broad sense to refer to atleast a reduction in the magnitude of a parameter, e.g., symptom,associated with the pathological condition being treated, such asdisease or disorder associated with (e.g., caused by) the target cell orpopulation thereof, where the editing of the surface of the target cellis beneficial. As such, treatment also includes situations where thepathological condition, or at least symptoms associated therewith, arecompletely inhibited, e.g., prevented from happening, or stopped, e.g.terminated, such that the individual no longer suffers from thepathological condition, or at least the symptoms that characterize thepathological condition.

Dosing is dependent on severity and responsiveness of the disease stateto be treated. Optimal dosing schedules can be calculated frommeasurements of conjugate accumulation in the body of the individual.The administering physician can determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of conjugate, and can generally be estimatedbased on EC₅₀s found to be effective in in vitro and in vivo animalmodels, etc. In general, dosage is from 0.01 μg to 100 g per kg of bodyweight, and may be given once or more daily, weekly, monthly or yearly.The treating physician can estimate repetition rates for dosing based onmeasured residence times and concentrations of the drug in bodily fluidsor tissues. Following successful treatment, it may be desirable to havethe subject undergo maintenance therapy to prevent the recurrence of thedisease state, where the conjugate is administered in maintenance doses,once or more daily, to once every several months, once every six months,once every year, or at any other suitable frequency.

The therapeutic methods of the present disclosure may includeadministering a single type of conjugate to an individual, or mayinclude administering two or more types of conjugates to an individual(e.g., a cocktail of different conjugates), where the two or more typesof conjugates may be designed to edit the surface of the same type ordifferent types of target cells.

In certain aspects, a conjugate of the present disclosure isadministered to the individual in combination with a second therapeuticagent as part of a combination therapy. Such administration may includeadministering the conjugate and the second agent concurrently, oradministering the conjugate and the second agent sequentially. In someembodiments, the individual has cancer, and the second therapeutic agentis an anti-cancer agent. Anti-cancer agents of interest include, but arenot limited to, anti-cancer antibodies (e.g., any of the antibodies setforth in Tables 1, 2, and 3 above), small molecule anti-cancer agents,or the like.

In some embodiments, the second therapeutic agent is a small moleculeanti-cancer agent selected from abiraterone, bendamustine, bexarotene,bortezomib, clofarabine, decitabine, exemestane, temozolomide, afatinib,axitinib, bosutinib, cabozantinib, crizotinib, dabrafenib, dasatinib,erlotinib, gefitinib, ibrutinib, imatinib, lapatinib, nilotinib,pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib, sunitinib,vandetanib, vemurafenib, enzalutamide, fulvestrant, epirubicin,ixabepilone, nelarabine, vismodegib, cabazitaxel, pemetrexed,azacitidine, carfilzomib, everolimus, temsirolimus, eribulin,omacetaxine, trametinib, lenalidomide, pomalidomide, romidepsin,vorinostat, brigatinib, ribociclib, midostaurin, telotristat ethyl,niraparib, cabozantinib, lenvatinib, rucaparib, granisetron, dronabinol,venetoclax, alectinib, cobimetinib, panobinostat, palbociclib,talimogene laherparepvec, lenvatinib, trifluridine and tipiracil,ixazomib, sonidegib, osimertinib, rolapitant, uridine triacetate,trabectedin, netupitant and palonosetron, belinostat, ibrutinib,olaparib, idelalisib, and ceritinib.

In certain aspects, the second therapeutic agent is an immune checkpointinhibitor. Immune checkpoint inhibitors of interest include, but are notlimited to, inhibitors (e.g., antibodies) that target PD-1, PD-L1,CTLA-4, TIM3, LAGS, or a member of the B7 family. According to certainembodiments, the conjugate and the second therapeutic agent areadministered according to a dosing regimen approved for individual use.In some embodiments, the administration of the second therapeutic agentpermits the conjugate administered to the individual to be administeredaccording to a dosing regimen that involves one or more lower and/orless frequent doses, and/or a reduced number of cycles as compared withthat utilized when the conjugate is administered without administrationof the second therapeutic agent. In certain aspects, the administrationof the conjugate permits the second therapeutic agent administered tothe individual to be administered according to a dosing regimen thatinvolves one or more lower and/or less frequent doses, and/or a reducednumber of cycles as compared with that utilized when the secondtherapeutic agent is administered without administration of theconjugate.

In certain aspects, desired relative dosing regimens for agentsadministered in combination may be assessed or determined empirically,for example using ex vivo, in vivo and/or in vitro models; in someembodiments, such assessment or empirical determination is made in vivo,in a patient population (e.g., so that a correlation is established), oralternatively in a particular individual of interest.

In certain aspects, one or more doses of the conjugate and the secondtherapeutic agent are administered to the individual at the same time;in some such embodiments, such agents may be administered present in thesame pharmaceutical composition. In some embodiments, however, theconjugate and the second therapeutic agent are administered to theindividual in different compositions and/or at different times. Forexample, the conjugate may be administered prior to administration ofthe second therapeutic agent (e.g., in a particular cycle).Alternatively, the second therapeutic agent may be administered prior toadministration of the conjugate (e.g., in a particular cycle). Thesecond agent to be administered may be administered a period of timethat starts at least 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 48hours, 72 hours, or up to 5 days or more after the administration of thefirst agent to be administered.

Kits

As summarize above, the present disclosure provides kits. According tocertain embodiments, the kits include any of the conjugates orcompositions of the present disclosure. The kits find use, e.g., inpracticing the methods of the present disclosure. For example, kits forpracticing the subject methods may include a quantity of thecompositions of the present disclosure, present in unit dosages, e.g.,ampoules, or a multi-dosage format. As such, in certain embodiments, thekits may include one or more (e.g., two or more) unit dosages (e.g.,ampoules) of a composition that includes a conjugate of the presentdisclosure. The term “unit dosage”, as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of thecomposition calculated in an amount sufficient to produce the desiredeffect. The amount of the unit dosage depends on various factors, suchas the particular conjugate employed, the effect to be achieved, and thepharmacodynamics associated with the conjugate in the subject. In yetother embodiments, the kits may include a single multi dosage amount ofthe composition.

Components of the kits may be present in separate containers, ormultiple components may be present in a single container. A suitablecontainer includes a single tube (e.g., vial), one or more wells of aplate (e.g., a 96-well plate, a 384-well plate, etc.), or the like.

According to certain embodiments, a kit of the present disclosureincludes instructions for using the composition to treat an individualin need thereof. The instructions may be recorded on a suitablerecording medium. For example, the instructions may be printed on asubstrate, such as paper or plastic, etc. As such, the instructions maybe present in the kits as a package insert, in the labeling of thecontainer of the kit or components thereof (i.e., associated with thepackaging or sub-packaging) etc. In other embodiments, the instructionsare present as an electronic storage data file present on a suitablecomputer readable storage medium, e.g., portable flash drive, DVD,CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, the means for obtaining theinstructions is recorded on a suitable substrate.

The following examples are offered by way of illustration and not by wayof limitation.

Experimental Materials and Methods

PBS buffer, DPBS buffer, DMEM, RPMI-1640 media and heat-inactivatedfetal bovine serum were obtained from Corning-Mediatech. X-VIVO 15serum-free medium was purchased from Lonza. LB agar, 2xYT andAntibiotic-Antimycotic were purchased from Fisher Scientific and 4-12%Bis-Tris gels for SDS-PAGE were purchased from Bio-Rad.

Heat-inactivated human male AB serum was purchased from Sigma-Aldrich.Human recombinant IL-2, human recombinant IL-4, and human recombinantIL-13 were purchased from Biolegend. Humanized anti-Her2-IgG with analdehyde tag was a gift from Catalent Pharma Solutions (Emeryville,Calif.). Absorbance spectra were measured with a SpectraMax i3x(Molecular Devices). Pierce High-Capacity Endotoxin Removal SpinColumns, Pierce

LAL Chromogenic Endotoxin Quantitation Kit and LDH cytotoxicity assaykit were obtained from Thermo Fisher Scientific.Bicyclononyne-N-hydroxysuccinimide ester (BCN-NHS) andaminooxy-tetraethyleneglycol-azide (aminooxy-TEG-N₃) were purchased fromBerry & Associates, Inc.Dibenzocyclooctyne-tetrapolyethyleneglycol-maleimide(DBCO-PEG4-Maleimide) was purchased from Click Chemistry Tools.2′-(4-Methylumbelliferyl)-α-D-N-acetylneuraminic acid (MuNeuNAc) wasobtained from Biosynth International Inc. All other chemicals werepurchased from Sigma-Aldrich and used without further purification.

The following antibodies and recombinant proteins were used: Humanrecombinant Siglec-7-Fc chimera, Siglec-9-Fc chimera, NKG2D-Fc chimeraproteins, AF488-labeled anti-Siglec-7 mAb (clone 194211) and blockinganti-NKG2D mAb (clone 149810) were purchased from R&D Systems.Fluorescein isothiocyanate (FITC)-labeled Sambucus nigra (SNA) lectinwas obtained from EY Laboratories. AF647-labeled anti-Her2 mAb (clone24D2), AF647-labeled anti-CD16 mAb (clone 3G8), AF647-labeled anti-CD56mAb (clone HCD56), blocking anti-Siglec-7 mAb (clone S7.7), blockinganti-Siglec-9 mAb (clone K8) were obtained from Biolegend. TRITC-labeledanti-Fc mAb was purchased from Jackson lmmunoresearch. FITC-labeledanti-CD3 mAb (clone BW264/56) was purchased from Miltenyi Biotec.Humanized anti-Her2-IgG with C-terminal aldehyde-tag was a gift fromCatalent Pharma Solutions (Emeryville, Calif.).

Cell Lines and Cell Culture

Breast cancer cells SKBR3, HCC-1954, MDA-MB-453, ZR-75-1, BT-20,MDA-MB-231, and MDA-MB-468 were obtained from American Type CultureCollection (ATCC). SKBR3, HCC-1954, ZR-75-1, and MDA-MB-468 weremaintained in RPMI-1640 medium supplemented with 10% heat-inactivatedfetal bovine serum, plus 0.4% Antibiotic-Antimycotic and L-glutamine(300 mg/L). MDA-MB-453, BT-20, and MDA-MB-231 were maintained in DMEMmedium supplemented with 10% heat-inactivated fetal bovine serum, plus0.4% Antibiotic-Antimycotic, L-glucose (4.5 g/L), L-glutamine (584 mg/L)and sodium pyruvate (110 mg/L).

Peripheral blood mononuclear cells (PBMCs) were obtained from healthyblood bank donors and were isolated using Ficoll-Paque (GE HealthcareLife Sciences, GE-17-1440-02) density gradient separation. NK cells wereisolated from PBMCs by negative selection using the MACS NK cellisolation kit (Miltenyi Biotec, 130-092-657) and LS columns (MiltenyiBiotec, 130-042-401) according to the manufacturer's protocol andcultured in X-VIVO 15 supplemented with 5% heat-inactivated human maleAB serum (Sigma-Aldrich), and 100 ng/mL recombinant human interleukin-2(IL-2) (Biolegend) overnight before using.

NK cell enrichment was verified by flow cytometry to result in >95%CD56+/CD3− cells (see, FIG. 16). Monocytes were isolated from PBMCsusing the Pan Monocyte isolation kit (Miltenyi Biotec 130-096-537).CD16+ monocytes were isolated from PBMCs using the CD16+ Monocyteisolation kit (Miltenyi Biotec 130-091-765). After isolating freshPBMCs, M1 and M2-polarized macrophages were acquired by first platingfreshly isolated PBMCs in serum-free RPMI a T75 flask (Fisher Scientific1368065) at 37° C. in 5% CO₂ for 2 hours, then removing media andwashing cells three times with phosphate buffered saline (PBS+Ca+Mg) toisolate monocytes. M1-polarized cells were generated by incubatingremaining monocytes with 50 ng/mL recombinant human GM-CSF (PeproTech300-03) for 6 days in RPMI+20% heat inactivated fetal bovine serum,followed by 4 days incubation with 100 ng/mL bacterialLipopolysaccharide (Invivogen tlrl-3pelps) and 20 ng/mL recombinanthuman IFNγ (PeproTech 300-02BC) in RPMI with 10% heat-inactivated fetalbovine serum. M2-polarized macrophages were generated by incubatingmonocytes with 50 ng/mL recombinant human M-CSF (PeproTech 300-25) for 6days in RPMI+20% heat inactivated fetal bovine serum followed by 4 daysincubation with 20 ng/mL recombinant human IL-13 (carrier-free)(Biolegend 571102) and 100 ng/mL recombinant human IL-4 (carrier-free)(Biolegend 574004). Human γδ T cells were isolated from PBMCs bynegative selection with the EasySep™ Human Gamma/Delta T Cell IsolationKit (Stemcell Tech 19255).

FAGS Analysis

Cells were incubated with sialidase, anti-Her2-IgG, anti-Her2-IgG-Sia,or PBS control for 1 hour at 37° C. After three washes with PBS, cellswere resuspended in cold PBS with 0.5% bovine serum albumin (BSA)containing the probe of choice: antibody, receptor-Fc fusion proteinwith secondary anti-Fc antibody pre-complexed in solution, orFITC-labeled SNA lectin. Cells and antibodies/fusion proteins wereincubated for 30 mins at 4° C. in the dark. After three washes with PBSwith 0.5% BSA, the cells were brought up in PBS with 0.5% BSA thenanalyzed by flow cytometry. All flow cytometry data was analyzed usingFlowJo v. 10.0 (Tree Star).

Expression and Purification of Sialidases

Escherichia coli C600 transformed with plasmid pCVD364 containing theVibrio cholerae sialidase gene was a gift from Prof. Eric R. Vimr,University of Illinois, Urbana-Champaign. Cells were grown in 2xYTmedia, supplemented with ampicillin (100 μg/mL) at 37° C. for 12 hours.After incubation, the cells were harvested by centrifugation at 4, 700×gfor 10 min. And the pellet was resuspended in osmotic shock buffer (20%sucrose, 1 mM EDTA, 30 mM Tris-HCl, pH 8.0) and shaken gently for 10 minat room temperature. The cells were collected by centrifugation (9,000×gfor 10 min) and the pellets were resuspended in ice-cold pure water.After a 10 min incubation at 4° C., the supernatant was obtained bycentrifugation at 9,000×g for 10 min. To purify the protein, the samplewas further concentrated using an Amicon ultrafiltration device(membrane molecular mass cutoff, 30, 000 Da), reconstituted in 0.02 MTris-HCl buffer (pH 7.6), and loaded onto a HitrapQ-HP anion-exchangecolumn (GE Healthcare Life Sciences, 17-1154-01). The protein was elutedwith a gradient of NaCl in 0.02 M Tris-HCl buffer (pH 7.6) at a flowrate of 5 mL/min. The protein fractions with expected molecular mass asdetermined by SDS-PAGE stained with Coomassie brilliant blue werecollected and pooled. Endotoxins were removed using high-capacityendotoxin removal spin kit (Thermo Fisher Scientific, 88275) and theendotoxin concentration of the sample was determined by LAL chromogenicendotoxin quantitation kit (Thermo Fisher Scientific, 88282).

The Salmonella typhimurium sialidase gene was cloned into a pET151vector with an N-terminal Hexahisitidine tag and C-terminal aldehyde tag(SLCTPSRGS) and transformed into BL21(DE3) competent E. coli (NEBC2527H). Cells were grown in 2xYT media, supplemented with ampicillin(100 μg/mL) at 37° C. for until they reached an optical density of 0.6,then 0.3 mM IPTG was added and the cells were grown at 37° C. shakingfor 16 hours. After incubation, the cells were harvested bycentrifugation at 4, 700×g for 10 min. And the pellet was resuspended in50 mL lysis buffer (phosphate buffered saline

(Fisher Scientific MT21040cv)+150 mM NaCl+10 mM imidazole. A proteaseinhibitor tablet (Sigma 5892970001) and 1 μL of nuclease (ThermoScientific-Pierce 88702) was added and the cells in lysis buffer wereincubated at 4° C. shaking for 2 hours. Cells were lysed via homogenizerand purified using nickel-NTA resin (Thermo Fisher 88221) with 250 mMimidazole elution. The protein fractions with expected molecular mass asdetermined by SDS-PAGE stained with Coomassie brilliant blue werecollected and pooled. Endotoxins were removed using high-capacityendotoxin removal spin kit (Thermo Fisher Scientific, 88275) and theendotoxin concentration of the sample was determined by LAL chromogenicendotoxin quantitation kit (Thermo Fisher Scientific, 88282).

Activity Assay of Sialidases using MuNeuNAc

5 μL of sialidase (30-60 nM in DPBS buffer with Ca²⁺ and Mg²⁺, pH 7.0)was added to 50 μL solution containing 0.1 mM2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MuNeuNAc,Biosynth International Inc.) in DPBS buffer with Ca^(2') and Mg²⁺ (pH7.0). After incubation for 10 min at 37° C., the mixture was dilutedwith 150 μL of 0.1 M glycine-NaOH buffer, pH 10.4. Fluorescence was readwith a fluorescence spectrophotometer (excitation 360 nm; emission 440nm). Activity is reported as U/mg, where a unit is defined as the amountof enzyme required to release 1 μmol of methylumbelliferone per minutein DPBS buffer, pH 7.

Preparation of Anti-Her2-IgG-Sia

Purified Vibrio cholerae sialidase (2 mg/mL in DPBS buffer with Ca²⁺ andMg²⁺, pH 7.0) was reacted with 12 equivalent ofbicyclononyne-N-hydroxysuccinimide ester (BCN-NHS) at 4° C. overnight.Excess linker was removed using a PD-10 Desalting Column (GE HealthcareLife Sciences, 17-0851-01). The degree of labeling was determined byESI-MS (see, FIG. 6B). Humanized anti-Her2-IgG with C-terminalaldehyde-tag was produced as described previously. Anti-Her2-IgG-Sia wasprepared by first coupling anti-Her2-IgG with C-terminal aldehyde-tag(120 μM) to aminooxy-tetraethyleneglycol-azide (aminooxy-TEG-N₃) (10 mM)in 100 mM ammonium acetate buffer, pH 4.5, at 37° C. for 10 days,followed by buffer-exchange into DPBS buffer with Ca²⁺ and Mg²⁺ (pH 7.0)using a PD-10 Desalting Column (GE Healthcare Life Sciences,17-0851-01). The resulting conjugate was then coupled to labeledsialidase at 1:28 molar ratio at 120 mg/mL total protein concentrationin DPBS buffer with Ca²⁺ and Mg²⁺ (pH 7.0). After a 3 day incubation atroom temperature, anti-Her2-IgG-Sia was purified by size exclusionchromatography Superdex 200. The purified product was analyzed bySDS-PAGE gel and ESI-MS.

Purified Salmonella typhimurium sialidase (3 mg/mL in DPBS buffer withCa²⁺ and Mg²⁺, pH 7.0) was reacted with 20 equivalent ofDBCO-PEG4-Maleimide at 4° C. overnight. Excess linker was removed usinga PD-10 desalting column (GE Healthcare Life Sciences, 17-0851-01). Thedegree of labeling was determined by ESI-MS (see FIG. 6B). Humanizedanti-Her2-IgG with C-terminal aldehyde-tag was produced as describedpreviously. Anti-Her2-IgG-Sia was prepared by first couplinganti-Her2-IgG with C-terminal aldehyde-tag (120 μM) toaminooxy-tetraethyleneglycol-azide (aminooxy-TEG-N₃) (10 mM) in 100 mMammonium acetate buffer, pH 4.5, at 37° C. for 10 days, followed bybuffer-exchange into

DPBS buffer with Ca²⁺ and Mg²⁺ (pH 7.0) using a PD-10 Desalting Column(GE Healthcare Life Sciences, 17-0851-01). The resulting conjugate wasthen coupled to labeled sialidase at 1:14 molar ratio at 25 mg/mL totalprotein concentration in DPBS buffer with Ca²⁺ and Mg²⁺ (pH 7.0). Aftera 3 day incubation at room temperature, anti-Her2-IgG-Sia was purifiedby size exclusion chromatography Superdex 200. The purified product wasanalyzed by SDS-PAGE gel and ESI-MS.

Cell Cytotoxicity Assay

Antibody-dependent cellular cytotoxicity (ADCC) was analyzed bymeasuring lactate dehydrogenase (LDH) release from breast cancer cellsas a result of ADCC activity of peripheral blood mononuclear cells(PBMCs), NK cells, monocytes, CD16+ monocytes, M1 macrophages, or M2macrophages. Tumor cells (target cells) were co-incubated with PBMCs, NKcells, monocytes, or macrophages (effector cells) at variouseffector/target (E/T) ratios in the presence or absence of sialidase ormAbs in triplicate. In a typical experiment, 100 μL of effector cellswere added to a V-bottom 96-well plate containing 100 μL of target cellsat 2×10⁵ cells/mL. After 4 hours, supernatants were collected, and LDHrelease was measured using a LDH cytotoxicity assay kit (Thermo FisherScientific, 88954) according to the manufacturer's protocol. Theabsorbance at 490 nm was measured with a SpectraMax i3x (MolecularDevices). Specific lysis was calculated as 100×(experimental−effectorcells spontaneous release−target cells spontaneous release)/(targetcells maximum release−target cells spontaneous release).

Fluorescence Microscopy

For visualization of HER2-specific enzymatic activity of the conjugate:cells were incubated with various concentrations of anti-Her2-IgG-Sia inPBS buffer for 1 hour at 37° C. After washes with PBS, cells were thenfixed with 4% formaldehyde at room temperature for 20 min. The fixedcells were washed with 0.5% BSA in PBS three times, followed by blockingin PBS with 0.5% BSA for 1 hour. Cells were incubated with FITC-labeledSNA (1:100) and AF647-labeled anti-Her2 antibody (1:100) in 0.5% BSA inPBS for 30 min at room temperature in the dark with gentle shaking.After washing thrice with 0.5% BSA in PBS, DAPI (1:1250 dilution from a10 mM stock) was added right before imaging with a Nikon A1R+ ResonantScanning Confocal Microscope.

For visualization of NK-tumor cell synapses: tumor cells were incubatedwith 6 nM anti-Her2-IgG or 6 nM anti-Her2-IgG-Sia in PBS buffer for 1hour at 37° C. Freshly isolated NK cells were added to tumor cells at anE/T ratio of 2:1 and incubated together for 15 min at 37° C. Afterwashing with PBS, cells were fixed with 4% formaldehyde in PBS for 20min at room temperature. The fixed cells were washed with 0.5% BSA inPBS three times, followed by blocking in PBS with 0.5% BSA for 1 hour.Cells were incubated with a mixture of AF488-labeled anti-Siglec 7(1:100), TRITC-labeled anti-Fc (1:400), and AF647-labeled anti-CD16(1:100) in PBS buffer for 30 min at room temperature in the dark withgentle shaking. After washing thrice with 0.5% BSA in PBS, DAPI (1:1250dilution from a 10 mM stock) was added right before imaging with a NikonAl R+Resonant Scanning Confocal Microscope.

Statistical Analysis

Statistical analyses were conducted with Prism 6. Data are shown asmean±SD of triplicate experiments, and significance was determined usinga t-test, unless otherwise noted. **=p<0.005, *=p<0.05, and a pvalue>0.05 was considered significant.

Introduction

When sufficiently abundant, glycans terminating in sialic acid residuescreate a signature of “healthy self” that suppresses immune activationvia several pathways—through recruitment of complement factor H andsubsequent down-regulation of the alternative complement cascade, forexample, and by recruitment of immunosuppressive sialic acid-bindingIg-like lectins (Siglecs) found on most types of leukocytes to theimmunological synapse. Sialylation status plays an important role in acell's ability to trigger or evade immunological recognition.

Upregulation of sialylated glycans has been correlated with poorprognosis and decreased immunogenicity of tumors. Hypersialylation ofcancer cells may contribute to evasion of immune surveillance by NKcells, the major mediators of antibody-dependent cell-mediatedcytotoxicity (ADCC). Dense populations of sialylated glycans can recruitNK cell-associated Siglec-7 and/or Siglec-9 to the immune synapse (FIG.1). Like PD-1, these

Siglecs possess a cytosolic immunoreceptor tyrosine-based inhibitory(ITIM) motif that mediates suppression of signals from activating NKcell receptors (FIG. 1). Engineered hypersialylation of tumor targets isprotective from innate NK cell killing as well as ADCC in aSiglec-7-dependent manner. Likewise, enzymatic removal of sialic acidsby treatment of tumor cells with sialidase potentiates NK cell-mediatedkilling, as does inhibition of Siglec-7 or -9 with blocking antibodies.Sialylation of cancer cell glycans also disrupts the interaction of theNK-activating receptor, natural killer group 2D (NKG2D), with itscognate ligands, thus reducing NK-activating signals from tumor cells(FIG. 1). Conversely, removal of cell-surface sialic acids enhances NKcell activation by increasing NKG2D-ligand binding. Thus, during themicroevolutionary process of tumor progression, hypersialylationprovides a selective advantage by reducing NK activating signals whileenhancing NK inhibitory signals emanating from the immune synapse.

An immune evasion strategy targeting NK-activating receptors andNK-inhibitory receptors using sialic acids is schematically illustratedin FIG. 1. In sialic acid-overexpressing cancer cells, hypersialylatedglycans interact with NK inhibitory receptors, leading to inhibition ofNK cells activation. Removal of cell-surface sialic acids byantibody-sialidase conjugate abolishes the interaction of sialylatedglycans and NK-inhibitory receptors, and increases the binding betweenNK-activating receptor and its ligands, thereby enhancing the tumor cellsusceptibility to NK cell-mediated ADCC.

It was reasoned that tumor-specific desialylation could be a powerfulintervention that potentiates tumor cytolysis by NK cells. It isreported here that an antibody-enzyme conjugate (AEC) can selectivelyedit the tumor cell glycocalyx and potentiate NK cell killing by ADCC, atherapeutically important mechanism harnessed by many antibody cancerdrugs. A recombinant sialidase was chemically fused to theHER2-targeting therapeutic monoclonal antibody trastuzumab. Theantibody-sialidase conjugate desialylated tumor cells in aHER2-dependent manner, destroyed ligands for inhibitory Siglecs whileenhancing NKG2D binding, and amplified NK cell killing compared totrastuzumab alone (FIG. 1).

Example 1—Suitability of V. cholera and S. typhimurium Sialidases

To identify suitable sialidases for the trastuzumab AEC, a panel ofenzymes were expressed and purified as described previously (FIG. 3A andB) and the Vibrio cholera and Salmonella typhimurium sialidases wereidentified as well suited for this purpose. V. cholerae and S.typhimurium sialidases were expressed and purified as describedpreviously. The purity of protein was determined by SDS-PAGE gel andESI-MS (FIG. 2B, 2E, and FIG. 3B, and FIG. 7A, 7F). Approximately 15 mgof enzymes were purified from 1 liter of cultured cells, with an invitro hydrolytic activity of more than 10 U/mg for V. cholerae and 114for S. typhimurium as measured with the fluorogenic substrate2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MuNeuNAc) aspreviously reported, where a unit is defined as the amount of enzymerequired to release 1 μmol of methylumbelliferone per minute in DPBSbuffer, pH 7. To determine if V. cholerae sialidase could efficientlyremove sialic acids from cell-surface glycans, its effects on cellsurface labeling was tested with FITC-labeled Sambucus nigra agglutinin(SNA). As well, the effects of V. cholerae treatment on cell labelingwere evaluated with receptor-Fc chimeras comprising the ectodomains ofSiglec-7, Siglec-9 or NKG2D. Desialylation of various tumor cell linesby sialidase at 37° C. for 1 hour significantly reduced binding of SNAas well as Siglec-7-Fc and Siglec-9-Fc chimeras (FIG. 4). With adecrease in SNA binding, an increase in binding capacity of NKG2D-Fcchimera was observed for most breast cancer cell lines after sialidasetreatment (FIG. 4D).

Preparation and characterization of antibody-sialidase conjugates isshown in FIG. 2. FIG. 2A schematically illustrates the preparation ofantibody-vibrio cholerae sialidase conjugates. FIG. 2B shows SDS-PAGEanalysis of sialidase, trastuzumab, and sialidase-trastuzumab conjugateunder non-reducing (lanes 3, 4, and 5) and reducing conditions (lanes 6,7, and 8), visualized by coomassie staining. Pre-stained protein ladder:lanes 1, 2, and 9. FIG. 2C shows ESI-MS of antibody sialidase conjugatewith Vibrio cholerae sialidase. FIG. 2D schematically illustrates thepreparation of antibody-Salmonella typhimurium sialidase conjugates.FIG. 2E shows SDS-PAGE analysis of sialidase, DBCO-modified sialidase,trastuzumab, and trastuzumab-sialidase conjugate under non-reducingconditions (lanes 3, 4, 5, and 6) and trastuzumab andtrastuzumab-sialidase conjugate under reducing conditions (lanes 7 and8), visualized by coomassie staining. Pre-stained protein ladder: lanes1, 2, and 9. FIG. 2F shows ESI-MS of antibody-sialidase conjugate withSalmonella typhimurium sialidase.

FIG. 3 shows the characterization of a panel of sialidases. FIG. 3Adepicts activity of sialidases on the substrate2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MuNeuNAc).SDS-PAGE analysis of wild-type human neuraminidase 2, humanneuraminidase 3, V. cholerae sialidase, S. typhimurium sialidase, C.perfringens sialidase, and A. ureafaciens sialidase is shown in FIG. 3B,visualized by coomassie staining. FIG. 3C shows flow cytometry of Siglec9 ligand cleavage by V. cholerae, S. typhimurium, and humanNeuraminidase 2 from ZR-75-1 breast cancer cells. Siglec-7 ligands onBT-20 cells are efficiently removed after treatment with V. choleraesialidase, as shown in FIG. 3D.

Analysis of cell-surface sialylation levels of different breast cancercell lines with or without sialidase treatment is shown in FIG. 4A.Ligand levels of Siglec-7 on different breast cancer cell lines with orwithout sialidase treatment is shown in FIG. 4B. Ligand levels ofSiglec-9 on different breast cancer cell lines with or without sialidasetreatment is shown in FIG. 4C. Ligand levels of NKG2D on differentbreast cancer cell lines with or without sialidase treatment is shown inFIG. 4D.

Example 2—Removal of Cell-Surface Sialic Acids Enhance Susceptibility toADCC

To demonstrate that removal of cell-surface sialic acids can enhancetheir susceptibility to NK cell-mediated ADCC, ADCC assays wereperformed with SKBR3 (HER2 3+), MDA-MB-453 (HER2 2+) and BT-20 (HER2 1+)cell lines with and without the sialidase treatment in the presence of30 nM trastuzumab using purified human peripheral blood NK cells. Anapproximate 5%-100% increase in maximal cell killing was observed intrastuzumab-directed ADCC with various sialidase-treated cell lines(FIG. 5). To validate that the enhanced ADCC was due to sialidaseenzymatic activity, the hydrolytic activity assay and ADCC assay using aheat-inactivated V. cholerae sialidase was also performed. Inactivationof V. cholerae sialidase by heating to 80° C. for 20 minutes led to theloss of hydrolytic activity against sialic acid containing glycans aswell as the loss of the enhancement in ADCC (FIG. 6). It was expectedthat by conjugating sialidase to trastuzumab, increased localconcentration of sialidase on the cell-surface would provideproximity-enhanced activity and further potentiate the effect as well aslimit the promiscuity of the enzymatic activity in a tissue-specificmanner.

Shown in FIG. 5A is cytotoxicity of isolated peripheral blood NK cellsfrom healthy donors against BT-20 breast cancer cells alone (notreatment), in the presence of anti-Her2-IgG (Tras) or in the presenceof anti-HER2-IgG and human neuriminidase 2 (Neu2), human neuriminidase 3(Neu3), Vibrio cholerae sialidase (VCSia), Salmonella typhimuriumsialidase (STSia), Arthrobacter ureafaciens sialidase (AUSia), orClostridium perfringens sialidase, (CPSia). FIG. 5B depicts cytotoxicityof isolated peripheral blood NK cells from healthy donors againstdifferent breast cancer cells in the absence or presence of sialidase(30 nM), anti-Her2-IgG (30 nM) or a mixture of sialidase (30 nM) andanti-Her2-IgG (30 nM) at E/T ratios of 2:1 and 4:1. *P<0.05, **P<0.005.

FIG. 6 shows the characterization of wild-type and heat-inactivatedVibrio cholerae sialidase. Cytotoxicity of isolated peripheral blood NKcells against BT-20 cells in the absence or presence of anti-Her2-IgG(30 nM), sialidase (30 nM), a mixture of anti-Her2-IgG (30 nM) andsialidase (30 nM), heat-inactivated sialidase (HI-Sialidase 30 nM), or amixture of anti-Her2-IgG (30 nM) and heat-inactivated sialidase (30 nM)at an E/T ratio of 4:1 is shown in FIG. 6A. Hydrolytic activities ofwild-type and heat-inactivated V. cholerae sialidase using2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MuNeuNAc) isshown in FIG. 6B. Levels of Sambucus nigra lectin (SNA) ligands on BT-20cells with or without 30 nM wild-type sialidase or heat-inactivatedsialidase treatment is shown in FIG. 6C. Levels of Siglec-7 ligands onBT-20 cells with or without 30 nM wild-type sialidase orheat-inactivated sialidase treatment is shown in FIG. 6D. Levels ofSiglec-9 ligands on BT-20 cells with or without 30 nM wild-typesialidase or heat-inactivated sialidase treatment is shown in FIG. 6E.Levels of NKG2D ligands on BT-20 cells with or without 30 nM wild-typesialidase or heat-inactivated sialidase treatment is shown in FIG. 6F.**P<0.005, NS: not significant.

Example 3—Preparation and Characterization of Antibody-SialidaseConjugates

A key concern in designing the sialidase-trastuzumab AEC was to identifya site for enzyme conjugation that would not undermine binding toFcγRIII (CD16), the interaction that initiates ADCC. Inspiration fromthe field of antibody-drug conjugates (ADCs) was taken where sites ofattachment have been tailored to avoid interference with immune effectorfunctions. Accordingly, sialidase was chosen to link near the C-terminusof trastuzumab's heavy chain, far from the C_(H)2 domain at whichFcγRIII binds. The aldehyde tag method for site-specific conjugation wasused based on precedents of its use in the assembly of protein-proteinchemical fusions as well as site-specific antibody-drug conjugates.Trastuzumab (anti-Her2-IgG) bearing a C-terminal aldehyde tag wasobtained as previously described. The functionalized antibody was firstcoupled to aminooxy-tetraethyleneglycol-azide (aminooxy-TEG-N₃) (FIG.2A). In parallel, sialidases were prepared. V. cholerae sialidase wasrandomly functionalized on lysine residues withbicyclononyne-N-hydroxysuccinimide ester (BCN-NHS). After an overnightreaction, excess linker was removed and the extent of BCN-NHSmodification of sialidase was determined by ESI-MS (FIG. 7B). Finally,trastuzumab adorned with the azide-functionalized linker was conjugatedto BCN-functionalized V. cholerae sialidase via copper-free clickchemistry (FIG. 2A). The desired conjugate was purified using asize-exclusion column and its apparent molecular weight(anti-Her2-IgG-Sia, ca. 312 kDa) was confirmed by SDS-PAGE (FIG. 2B).ESI-MS analysis confirmed that the sialidase was covalently linked tothe heavy chain of trastuzumab (FIG. 2C and FIG. 7E). Sialidase activityof the final AEC was evaluated using the fluorogenic substrate MuNeuNAc.More than 85% enzymatic activity remained after the chemical conjugationprocess (FIG. 8). Alternatively, S. typhimurium sialidase wassite-specifically conjugated to the cysteine on the C-terminal aldehydetag by reacting with DBCO-PEG4-Maleimide overnight; following thisexcess linker was removed and the conjugation of DBCO to S. typhimuriumsialidase was determined to be complete by ESI-MS (FIG. 7G). Finallytrastuzumab with azide linker was conjugated to DBCO-functionalized S.typhimurium sialidase via copper-free click chemistry (FIG. 2D). Thedesired conjugate was purified using a size-exclusion column and itsapparent molecular weight (anti HER2-IgG-StSia, ca 240 kDa) wasconfirmed by SDS-PAGE (FIG. 2E). ESI-MS analysis confirmed that thesialidase was covalently linked to the heavy chain of trastuzumab (FIG.2F and FIG. 7H). Sialidase activity of the final AEC was evaluated usingthe fluorogenic substrate MuNeuNAc. A slight increase in enzymaticactivity compared to free aldehyde-tagged sialidase resulted after thechemical conjugation process to the free cysteine on the C-terminal tag(FIG. 8).

FIG. 7 shows ESI-MS spectra of sialidase, anti-Her2-IgG and itsconjugates. ESI-MS spectrum of purified Vibrio cholerae sialidase isshown in FIG. 7A. ESI-MS spectrum of V. cholerae sialidase labeled withBCN-NHS at 1:12 molar ratio is shown in FIG. 7B. ESI-MS spectrum ofanti-Her2-IgG with C-terminal aldehyde tag is shown in FIG. 7C. ESI-MSspectrum of anti-Her2-IgG with C-terminal aldehyde tag conjugated withaminooxy-TEG-azide is shown in FIG. 7D. ESI-MS spectrum ofanti-Her2-IgG-Sia is shown in FIG. 7E. ESI-MS of purified Salmonellatyphimurium sialidase is shown in FIG. 7F. ESI-MS of S. typhimuriumlabeled with DBCO_PEG4-Maleimide at a 1:20 molar ratio is shown in FIG.7G. ESI-MS spectrum of anti-HER2-IgG-St-Sia

FIG. 8 shows the hydrolytic activities of V. cholerae sialidase andanti-Her2-IgG-Sia, as well as S. typhimurium sialidase andanti-Her2-IgG-StSia against substrate2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (MuNeuNAc)

To further demonstrate that anti-Her2-IgG-Sia is able to specificallyremove sialic acid on HER2-expressing cells, SKBR3 (HER2 3+) andMDA-MB-468 (HER2 0) were incubated in the absence or presence of 6 nM or60 nM anti-Her2-IgG-Sia. As shown in FIG. 9 and FIG. 10, treatment with6 nM anti-Her2-IgG-Sia for 1 h resulted in a selective desialylation ofSKBR3 cells even in the presence of MDA-MB-468 cells (FIG. 9). However,this effect is dose-dependent. Surface sialic acid levels of SKBR3 andMDA-MB-468 cells were both reduced with a treatment at 60 nM ofanti-Her2-IgG-Sia for 1 h. This effect was quantified using flowcytometry on mixtures of cells treated with various concentrations ofanti-Her2-IgG-Sia (FIG. 9A). However, in another conjugate with asmaller sialidase lacking lectin domains (anti-HER2-IgG-St-Sia) surfacesialic acid levels of off-target HER2 0 MDA-MB-468 cells remaineduntouched until much higher concentrations of about 1 μManti-Her2-IgG-St-Sia conjugate (FIG. 9B).

FIG. 9 shows in vitro characterization of trastuzumab andtrastuzumab-sialidase conjugate with different HER2-expressing cancercells. Cell-surface sialic acid on the

HER2-high expressing cell line, SKBR3, can be selectively removed using6 nM trastuzumab-sialidase conjugate. Scale bar, 25 μm.

FIG. 10 shows SNA ligands on SKBR3 and MDA-MB-468 cells in the absenceor presence of anti-Her2-IgG-Sia conjugate. SNA ligands on individualcultures of SKBR3 and MDA-MB-468 cells in the absence or presence ofanti-Her2-IgG-Sia conjugate is shown in FIG. 10A. Cells were incubatedwith 6 nM anti-Her2-IgG-Sia conjugate or PBS in RPMI-1640 media for 1hour at 37° C. and stained with FITC-labeled SNA lectin, AF647-labeledanti-Her2 and DAPI nuclear stain. SNA ligands on a mixture of SKBR3 andMDA-MB-468 cells in the absence or presence of anti-Her2-IgG-Siaconjugate is shown in FIG. 10B. SKBR3 and MDA-MB-468 cells were mixed ata 1:1 ratio and cultured overnight. The cell mixtures were incubated inthe absence or presence of 6 nM or 60 nM anti-Her2-IgG-Sia conjugate for1 hour at 37° C. Scale bar=25 μm.

To assess the effect of the antibody-sialidase conjugate on NKcell-mediated ADCC, cytotoxicity assays were performed using variousbreast cancer cell lines (SKBR3, HER2 3+; HCC-1954, HER2 3+; MDA-MB-453,HER2 2+; ZR-75-1, HER2 1+; BT-20, HER2 1+; MDA-MB-231, HER2 1+;MDA-MB-468, HER2 0) in the presence of anti-Her2-IgG oranti-Her2-IgG-Sia at effector/target (E/T) ratios of 4:1 and 8:1. Incomparison to anti-Her2-IgG, anti-Her2-IgG-Sia demonstrated increases of33% to 140% of maximal cell killing with HER2 1+ cell lines ZR-75-1,BT-20, and MDA-MB-231 (FIG. 11). In addition, BT-20 cells were exposedto purified human peripheral blood NK cells at various E/T ratios in theabsence or presence of sialidase (30 nM), anti-Her2-IgG (30 nM), oranti-Her2-IgG-Sia (30 nM) (FIG. 12A). Sialidase treatment alone of BT-20cells lines showed little NK cell-mediated cytotoxicity at different E/Tratios. Compared to anti-Her2-IgG, anti-Her2-IgG-Sia showedsignificantly improved cytolysis at various ratios. At an E/T ratio of4, the largest enhancement was observed: 46%±1 cytolysis foranti-Her2-IgG-Sia versus 21%±1 for anti-Her2-IgG. It was verified thatADCC was likely being mediated by NK cells as NK cell-depleted PBMCsshowed little cell lysis (FIG. 12B).

FIG. 11 shows cytotoxicity data of isolated peripheral blood NK cellsfrom healthy donors against different breast cancer cells in thepresence of anti-Her2-IgG (30 nM) or anti-Her2-IgG-Sia (30 nM) at E/Tratios of 4:1 and 8:1.

FIG. 12 shows in vitro activity of trastuzumab and trastuzumab-sialidaseconjugate against different HER2-expressing cancer cells. Cytotoxicityassays performed with BT-20 cells in the absence or presence ofsialidase (30 nM), anti-Her2-IgG (30 nM) and anti-Her2-IgG-Sia (30 nM)using NK cells are shown in FIG. 12A. Results of cytotoxicity assaysperformed with BT-20 cells in the absence or presence of sialidase (30nM), anti-Her2-IgG (30 nM) and anti-Her2-IgG-Sia (30 nM) using NKcells-depleted PBMCs are shown in FIG. 12B. The trend seen in theenhancement of ADCC correlated with Siglec-7-Fc, Siglec-9-Fc andNKG2D-Fc binding is shown in FIG. 12C-12F. Cytotoxic activity of NKcells against different HER2-expressing cancer cells in the presence ofindicated concentrations of trastuzumab and trastuzumab-sialidaseconjugate is shown in FIG. 12G-12J. Fluorescent microscopy analysis ofSiglec-7 distribution on NK cells with trastuzumab ortrastuzumab-sialidase conjugate treatments is shown in FIG. 12K.Siglec-7 displayed recruitment to the

NK synapse with trastuzumab treatment. After removing sialic acids onSKBR3 cells using trastuzumab-sialidase conjugate, Siglec-7 recruitmentto the NK-tumor synapse is lost. Scale bar, 10 μm, **P<0.005.

To assess the effect of the antibody-sialidase conjugate on cytotoxicitymediated by monocytes and macrophages, cytotoxicity assays wereperformed using breast cancer cell lines (SKBR3, HER2 3+; BT-20, HER21+) in the presence of Vibrio cholerae sialidase alone (VCSia),anti-Her2-IgG (Tras), or anti-Her2-IgG-Sia (T-Sia) at variouseffector/target (E/T) ratios. Whereas the total monocyte populationexhibited low overall killing of tumor cells, isolated CD16+ monocytesprimarily expressing Siglecs 3, 7, and 9 demonstrated increases of about100% upon treatment with the conjugate anti-Her2-IgG-Sia (T-Sia)compared to treatment with anti-Her2-IgG (Tras) (FIG. 13).Differentiated M1 macrophages expressing Siglecs 3, 6, 7, and 9, and M2macrophages expressing Siglecs 3, 5, 6, 7, 8, 9, 10, and 11 both appearto exhibit increases in cytotoxic killing of tumor cells withtrastuzumab-sialidase conjugate as opposed to trastuzumab alone (FIG.13). γδ T cell mediated cytotoxicity can also be potentiated bytrastuzumab sialidase conjugate (T-Sia) rather than treating withtrastuzumab (Tras) or sialidase (VCSia) alone (FIG. 14).

FIG. 13A depicts the siglec expression levels of a human isolatedmonocyte population as determined by flow cytometry. FIG. 13B showscytotoxicity data of isolated human monocytes against BT-20 breastcancer cells after 24 hours incubation with V. cholerae sialidase,anti-HER2-IgG (Tras), or anti-Her2-IgG-Sia (T-Sia) and an E:T ratio of1:8. FIG. 13C depicts the siglec receptor expression levels of CD16+monocytes isolated from the monocyte population. FIG. 13D. Showscytotoxicity data of CD16+ monocytes from healthy donors after fourhours incubation with V. cholerae sialidase, anti-HER2-IgG (Tras), oranti-Her2-IgG-Sia (T-Sia) and BT-20 breast cancer cells. FIG. 13Edepicts the siglec receptor expression levels of isolated monocytes fromhealthy donors that have been differentiated into M1 macrophages asdescribed previously. FIG. 13F shows cytotoxicity data from M1macrophages differentiated from isolated monocytes from healthy donorsafter 24 hours incubation with V. cholerae sialidase, anti-HER2-IgG(Tras), or anti-Her2-IgG-Sia (T-Sia) and SK-BR-3 breast cancer cells.FIG. 13G depicts the siglec receptor expression levels of isolatedmonocytes from healthy donors that have been differentiated into M2macrophages as described previously. FIG. 13H shows cytotoxicity datafrom M2 macrophages differentiated from isolated monocytes from healthydonors after 24 hours incubation with V. cholerae sialidase,anti-HER2-IgG (Tras), or anti-Her2-IgG-Sia (T-Sia) and SK-BR-3 breastcancer cells. FIG. 13I shows the CD16 expression level of the M2macrophages from (13E and 13F).

FIG. 14 depicts the cytotoxicity of isolated γδ T cells in the presenceof V. cholerae sialidase, anti-HER2-IgG (Tras), or anti-Her2-IgG-Sia(T-Sia) and SK-BIR-3 cells at an E:T of 5:1.

Example 4—Mechanisms of Enhanced ADCC Using Antibody-Sialidase Conjugate

Previous studies have suggested that hypersialylation of cancer cellsresults in the reduced binding of activating receptor NKG2D as well asenhanced binding of inhibitory receptors, Siglec-7 and Siglec-9, thusreducing NK-mediated cytotoxicity. To explore the mechanism of increasedADCC using trastuzumab-sialidase conjugate, fold increase of ADCC wascorrelated with receptor binding in various breast cancer cell lines.Cell lines with the highest increases of NK-mediated ADCC correlatedwith the highest levels of Siglec-7 and Siglec-9 binding (FIGS.12C-12E). Treatment of BT-20 and ZR-75-1 cells—those with the highestexpression of Siglec-7 and Siglec-9 surface ligands—withtrastuzumab-sialidase conjugate enhanced ADCC by more than 2 foldcompared to trastuzumab alone. In contrast, the conjugate offered littlesignificant improvement on ADCC of MDA-MB-453 cells, which have thelowest expression of Siglec-7 and Siglec-9 surface ligands. To furthersubstantiate that anti-Her2-IgG-Sia was enhancing ADCC through areduction in binding of inhibitory receptors Siglec-7 and Siglec-9 alongwith enhancement of the interaction with activating receptor NKG2D,blocking antibodies against Siglec-7, Siglec-9 and NKG2D were used tospecifically block ligand-receptor interactions. Anti-Siglec-7 andanti-Siglec-9 antibodies at 5 μg/mL led to significantly enhanced NKcell cytotoxicity against BT-20 cells with anti-Her2-IgG, but not with amixture of anti-Her2-IgG and sialidase (FIG. 15). In addition, blockingthe NKG2D receptor showed a greater effect on ADCC in the mixture ofanti-Her2-IgG and sialidase-treated cells compared toanti-Her2-IgG-mediated ADCC (FIG. 15).

FIG. 15 shows results relating to cytotoxicity of isolated peripheralblood NK cells from healthy donors against BT-20 cells withanti-Her2-IgG (30 nM) or a mixture of anti-Her2-IgG (30 nM) andsialidase (30 nM) in the absence or presence of 5 μg/mL blockinganti-NKG2D (clone 149810), anti-Siglec-7 (clone S7.7), anti-Siglec-9(clone S9), a mixture of anti-Siglec-7 (clone S7.7) and anti-Siglec-9(clone S9), or mouse IgG1 isotype antibody (clone MOPC-21) at an E/Tratio of 4:1. *P<0.05, **P<0.005, ns: not significant.

Example 5—Comparison Between Antibody-Sialidase Conjugate and AntibodyAlone

In order to directly compare the ability of anti-Her2-IgG-Sia to directADCC versus anti-Her2-IgG alone, the dose response for cytotoxicity wasmeasured using four different breast cancer cell lines: SKBR3 (HER2 3+),ZR-75-1 (HER2 1+), BT-20 (HER2 1+), MDA-MB-468 (HER2 0). Compared toanti-Her2-IgG, anti-Her2-IgG-Sia is more cytotoxic in all threeHER2-expressing cell lines at an E/T ratio of 4. For the HER2 3+ cellline, anti-Her2-IgG-Sia killed SKBR3 cells with an EC₅₀ of 76±14 pM,which was slightly better than anti-Her2-IgG (EC₅₀ 177±54 pM). While forHER2 1+ cell lines ZR-75-1 and BT-20, the anti-Her2-IgG-Sia is ˜10 timesmore cytotoxic than the anti-Her2-IgG (ZR-75-1 cells: anti-Her2-IgG-SiaEC₅₀ 135±47 pM, anti-Her2-IgG EC₅₀ 1143±274 pM; BT-20 cells:anti-Her2-IgG-Sia EC₅₀ 170±34 pM, anti-Her2-IgG EC₅₀ 1823±850 pM) (FIGS.12G-12J and Table 6). Little lysis of the HER2 negative cell lineMDA-MB-468 was observed for either anti-Her2-IgG or anti-Her2-IgG-Sia(FIG. 12J). Next, the difference between anti-Her2-IgG-Sia and a mixtureof anti-Her2-IgG and unconjugated sialidase (anti-Her2-IgG/sialidase)was tested. Anti-Her2-IgG-Sia showed lower EC₅₀ in SKBR3 cells(anti-Her2-IgG-Sia EC₅₀ 76±14 pM; anti-Her2-IgG/sialidase EC₅₀ 136±52pM), ZR-75-1 cells (anti-Her2-IgG-Sia EC₅₀ 135±47 pM;anti-Her2-IgG/sialidase EC₅₀ 492±67 pM), and BT-20 cells(anti-Her2-IgG-Sia EC₅₀ 170±34 μM; anti-Her2-IgG/sialidase EC₅₀ 692±156μM) (Table 6). The enhanced potency of the conjugate versus the mixtureof anti-Her2-IgG and unconjugated sialidase is evidence of a proximityeffect on the enzymatic activity.

TABLE 6 Cytotoxic activity of isolated NK cells against various humanbreast cancer cells induced by anti-Her2-IgG, a mixture of anti-Her2-IgGand sialidase, or anti-Her2-IgG-Sia conjugate. EC₅₀ (pM)/Maximal killing(%) Cell line HER2 level anti-Her2-IgG anti-Her2-IgG/sialidaseanti-Her2-IgG-Sia SKBR3 3+ 177 ± 54/61 ± 3 136 ± 52/64 ± 4 76 ± 14/66 ±2 HCC-1954 3+ 360 ± 67/46 ± 2 212 ± 50/46 ± 2 238 ± 41/49 ± 2 MDA-MB-4532+ 110 ± 27/71 ± 3 77 ± 17/78 ± 3 22 ± 5/75 ± 2 ZR-75-1 1+ 1143 ± 274/14± 1 492 ± 67/34 ± 1 135 ± 47/34 ± 2 BT-20 1+ 1823 ± 850/21 ± 1 692 ±156/51 ± 2 170 ± 34/46 ± 1 MDA-MB-231 1+ N.D./6 ± 1 N.D./10 ± 1 N.D./10± 1 MDA-MB-468 — N.D./2 ± 1 N.D./N.D. N.D./3 ± 1 (N.D. none detected).

Example 6—Sialidase Treatment Potentiates Rituximab-Induced CDC

B cell lymphoma cells, either Daudi or Ramos cell lines, were treatedwith sialidase or PBS control for 1 hour at 37° C. to desialylate theircell surfaces, then normal human serum (1:4, complete with complementproteins) and rituximab (10 μg/ml) was added and the mixture allowed toincubate for 2 hours at 37° C. The supernatant was collected and celldeath (cytotoxicity) was determined using a kit that measures LDHrelease from lysed cells then compared to fully detergent-lysed cells asa ‘100% killing’ standard. For FIG. 17: SiAse: sialidase treated cells,no rituximab. Rituxan: PBS treated and 10 μg/ml rituximab.SiaAse+Rituxan: sialidase treated and 10 μg/ml rituximab. *p<0.05

Daudi cells experience ˜10% increase in rituximab-inducedcomplement-dependent cytotoxicity (CDC). Ramos, on the other hand,experience almost twice as much CDC after desialylation.

Example 7—Ramos Cells Have Higher Levels of Siglec-9 Ligands than DaudiCells

The greater effect on CDC by desialylation seen with Ramos cells in thepreceding example could be explained by higher initial sialylation.

Siglec-Fc fusion proteins were pre-complexed at 5 μg/ml Sig-Fc and 4μg/mlanti-Fc secondary and incubated with cells for 30 min at 4° C.Cells were then washed 3 times and flow cytometry was performed.Separately, cells were treated with the anti-Fc secondary antibody (same4 μg/ml), then washed as above and flow cytometry was performed.Increase in fluorescence of the Siglec-Fc fusion treated cells over thesecondary-only treated cells indicates that binding was due to theSiglec-Fc protein, and not the secondary reagent.

Shown in FIG. 18 is the average +/−standard deviation of Siglec-9-Fcbinding of triplicate experimental replicates of Daudi and Ramos celllines. Ramos cells show ˜25% more Siglec-9 binding, thus likely displaymore sialic acid on their cell surfaces.

Example 8—Sialidase Potentiates Rituximab in a Complement-DependentManner

Complement protein C1q is a critical initiating component of the‘classical pathway’ of complement-dependent cytotoxicity. It helps formthe Cl complex, which binds antibodies on target cells and theninitiates the complement cascade which leads to cell death. As shown inFIG. 19, without C1q, the sialidase and/or rituximab does notefficiently lyse cells. This data indicates that sialidase treatment ispotentiating the rituximab-induced CDC via the classical pathway. Theseexperiments were performed as in FIG. 17, however for the red bars,serum that has been depleted of C1q was added in place of normal humanserum. The blue bars represent cells treated with normal human serum.*p<0.05, **p<0.01

Effect of Antibody-Sialidase Conjugate on the Immune Synapse

In previous work, it was demonstrated that Siglec-7 is recruited to theNK-target cell immunological synapse, thus inducing inhibitory signalingthrough an immunoreceptor tyrosine-based inhibition motif (ITIM). Toassess the effect of conjugated sialidase on the immune synapse, theimmune synapse was imaged during ADCC. SKBR3 cells were pre-incubatedwith anti-Her2-IgG or anti-Her2-IgG-Sia and then they were co-incubatedwith purified human peripheral blood NK cells to induce synapseformation. Cells were then fixed and stained for Siglec-7, HER2, FcγIII(CD16) and imaged by fluorescence microscopy. With anti-Her2-IgGtreatment, Siglec-7 co-localized with FcγIII (CD16) at the immunologicalsynapse formed with NK cells, which is consistent with its role as aninhibitory receptor of NK cell activation (FIG. 12K). In contrast, SKBR3cells treated with anti-Her2-IgG-Sia show little recruitment of Siglec-7despite an efficient recruitment of CD16. These results indicate thatthe trastuzumab-sialidase conjugate effectively remodels theimmune-cancer cell synapse while promoting ADCC.

Here, a new class of conjugates that are able to perform tissue-specificcell-surface glycan editing to enhance susceptibility to ADCC isreported. The conjugates provide the first means for a single antibodytherapy that simultaneously targets multiple immune-stimulatingpathways. Treatment of tumor cells with antibody-sialidase conjugate notonly actively recruits NK cells via Fc-FcγIII (CD16) interaction, butalso effectively retards the recruitment of inhibitory Siglec receptorsto the tumor-immune synapse and exposes activating NKG2D ligands throughprecise glycocalyx editing. Compared to trastuzumab treatment, the noveltrastuzumab-sialidase conjugate can efficiently direct NK cells to killHER2-expressing cancer cells and is even more efficient at targetingbreast cancer cells with antigens of low abundance. This has significantimplications for the ability to treat more moderate HER2-expressingtumors as trastuzumab is currently only prescribed to patients with thevery high HER2 expression levels.

Notably, macrophages and dendritic cells also express inhibitory Siglecs(Siglec-9 and -5, respectively). Thus, hypersialylation may be broadlyprotective against innate immune targeting by cells and complementfactors.

Unlike current cancer immune therapies, which each target a singlepathway, glycocalyx editing can affect multiple pathways across variousbranches of the immune system's armament.

Notwithstanding the appended claims, the present disclosure is alsodefined by the following clauses:

-   1. A conjugate, comprising:

a targeting moiety that binds to a cell surface molecule of a targetcell; and

a target cell surface-editing enzyme.

-   2. The conjugate of Clause 1, wherein the targeting moiety is    selected from the group consisting of: an antibody, a ligand, an    aptamer, a nanoparticle, and a small molecule.-   3. The conjugate of Clause 2, wherein the targeting moiety is an    antibody.-   4. The conjugate of Clause 3, wherein the antibody is an IgG, a    single chain Fv (scFv), Fab, (Fab)₂, or (scFv′)₂.-   5. The conjugate of Clause 3, wherein the antibody is an IgG1.-   6. The conjugate of any one of Clauses 3 to 5, wherein the antibody    is a monoclonal antibody.-   7. The conjugate of any one of Clauses 3 to 6, wherein the antibody    is a humanized or human antibody.-   8. The conjugate of any one of Clauses 3 to 7, wherein the target    cell surface-editing enzyme is conjugated to a light chain of the    antibody.-   9. The conjugate of any one of Clauses 3 to 7, wherein the target    cell surface-editing enzyme is conjugated to a heavy chain of the    antibody.-   10. The conjugate of Clause 9, wherein the target cell    surface-editing enzyme is conjugated to an Fc region of the    antibody.-   11. The conjugate of Clause 9, wherein the target cell    surface-editing enzyme is conjugated to the C-terminus of the heavy    chain.-   12. The conjugate of any one of Clauses 1 to 11, wherein the target    cell surface-editing enzyme is site-specifically conjugated to the    targeting moiety.-   13. The conjugate of Clause 12, wherein the targeting moiety    comprises a non-natural amino acid to which the target cell    surface-editing enzyme is site-specifically conjugated.-   14. The conjugate of any one of Clauses 1 to 13, wherein the target    cell surface-editing enzyme is conjugated to the targeting moiety    via a linker.-   15. The conjugate of Clause 14, wherein the linker comprises    polyethylene glycol (PEG).-   16. The conjugate of Clause 14, wherein the linker is a peptide.-   17. The conjugate of Clause 16, wherein the conjugate is a fusion    protein.-   18. The conjugate of any one of Clauses 1 to 15, wherein the target    cell is selected from the group consisting of: a cancer cell, an    immune cell, and an endothelial cell.-   19. The conjugate of Clause 18, wherein the target cell is a cancer    cell.-   20. The conjugate of Clause 19, wherein the cell surface molecule is    a tumor-associated cell surface molecule.-   21. The conjugate of Clause 19, wherein the cell surface molecule is    a tumor-specific cell surface molecule.-   22. The conjugate of any one of Clauses 19 to 21, wherein the cancer    cell is a carcinoma cell.-   23. The conjugate of any one of Clauses 19 to 22, wherein the cancer    cell is selected from the group consisting of: a breast cancer cell,    an ovarian cancer cell, a gastric cancer cell, and a colon cancer    cell.-   24. The conjugate of Clause 22 or Clause 23, wherein the cell    surface molecule is human epidermal growth factor receptor 2 (HER2).-   25. The conjugate of Clause 24, wherein the targeting moiety is    trastuzumab.-   26. The conjugate of any one of Clauses 3 to 18, wherein the    targeting moiety is selected from the group consisting of:    cetuximab, daratumumab, girentuximab, panitumumab, ofatumumab, and    rituximab.-   27. The conjugate of any one of Clauses 1 to 26, wherein the target    cell surface-editing enzyme cleaves a molecule on the surface of the    target cell, oxidizes a molecule on the surface of the target cell,    reduces a molecule on the surface of the target cell, adds a moiety    to a molecule on the surface of the target cell, or removes a moiety    from a molecule on the surface of the target cell.-   28. The conjugate of any one of Clauses 1 to 26, wherein the target    cell surface-editing enzyme cleaves a molecule on the surface of the    target cell.-   29. The conjugate of Clause 28, wherein the molecule on the surface    of the target cell is a ligand.-   30. The conjugate of Clause 29, wherein the ligand is a ligand of an    inhibitory immune receptor.-   31. The conjugate of Clause 30, wherein the inhibitory immune    receptor is present on an immune cell selected from the group    consisting of: a natural killer (NK) cell, a macrophage, a monocyte,    a neutrophil, a dendritic cell, a T cell, a B cell, a mast cell, a    basophil, and an eosinophil.-   32. The conjugate of Clause 31, wherein the inhibitory immune    receptor is a sialic acid-binding Ig-like lectin (Siglec) receptor.-   33. The conjugate of Clause 32, wherein the Siglec receptor is    Siglec 7.-   34. The conjugate of Clause 32, wherein the Siglec receptor is    Siglec 9.-   35. The conjugate of any one of Clauses 29 to 34, wherein the ligand    is a sialoglycan.-   36. The conjugate of any one of Clauses 1 to 35, wherein the target    cell surface-editing enzyme is a sialidase.-   37. The conjugate of Clause 36, wherein the sialidase is a    Salmonella typhimurium sialidase.-   38. The conjugate of Clause 36, wherein the sialidase is a Vibrio    cholerae sialidase.-   39. The conjugate of Clause 36, wherein the sialidase is a mammalian    neuraminidase.-   40. The conjugate of Clause 39, wherein the mammalian neuraminidase    is a human neuraminidase.-   41. The conjugate of Clause 40, wherein the human neuraminidase is    selected from the group consisting of: human neuraminidase 1, human    neuraminidase 2, human neuraminidase 3, and human neuraminidase 4.-   42. The conjugate of any one of Clauses 1 to 41, comprising two or    more target cell surface-editing enzymes conjugated to the targeting    moiety.-   43. A composition, comprising:

a conjugate of any one of Clauses 1 to 42; and a pharmaceuticallyacceptable carrier.

-   44. The composition of Clause 43, wherein the composition is    formulated for parenteral administration.-   45. A method comprising administering to an individual in need    thereof a conjugate of any one of Clauses 1 to 42 or a composition    of Clause 43 or Clause 44.-   46. A method of treating cancer comprising administering to an    individual having cancer a conjugate of any one of Clauses 1 to 42    or a composition of Clause 43 or Clause 44.-   47. A method of enhancing antibody-dependent cellular cytotoxicity    (ADCC) comprising administering to an individual in need of ADCC a    conjugate of any one of Clauses 1 to 39 or a composition of Clause    40 or Clause 41.-   48. The method according to any one of Clauses 45 to 47, wherein the    administering modulates an immune pathway in the individual.-   49. The method according to Clause 48, wherein the immune pathway is    selected from the group consisting of: an inhibitory immune receptor    pathway, a complement pathway, a paired immunoglobulin-like type 2    receptor (PILR) pathway, and a natural-killer group 2 member D    protein (NKG2D) pathway.-   50. The method according to any one of Clauses 45 to 49, wherein the    target cell comprises a ligand on its surface, and the administering    results in editing of the ligand by the target cell surface-editing    enzyme.-   51. The method according to Clause 50, wherein the editing of the    ligand comprises cleavage of all or a portion of the ligand.-   52. The method according to Clause 50 or Clause 51, wherein the    ligand is a ligand of an inhibitory immune receptor.-   53. The method according to Clause 52, wherein the inhibitory immune    receptor is present on an immune cell selected from the group    consisting of: a natural killer (NK) cell, a macrophage, a monocyte,    a neutrophil, a dendritic cell, a T cell, a B cell, a mast cell, a    basophil, and an eosinophil.-   54. The method according to Clause 53, wherein the inhibitory immune    receptor is a sialic acid-binding Ig-like lectin (Siglec) receptor.-   55. The method according to Clause 54, wherein the Siglec receptor    is Siglec 7.-   56. The method according to Clause 54, wherein the Siglec receptor    is Siglec 9.-   57. The method according to any one of Clauses 50 to 56, wherein the    ligand is a sialoglycan.-   58. The method according to Clause 57, wherein the target cell    surface-editing enzyme is a sialidase.-   59. The method according to Clause 58, wherein the sialidase is a    Salmonella typhimurium sialidase.-   60. The method according to Clause 58, wherein the sialidase is a    Vibrio cholerae sialidase.-   61. The method according to Clause 58, wherein the sialidase is a    mammalian neuraminidase.-   62. The method according to Clause 61, wherein the mammalian    neuraminidase is a human neuraminidase.-   63. The method according to Clause 62, wherein the human    neuraminidase is selected from the group consisting of: human    neuraminidase 1, human neuraminidase 2, human neuraminidase 3, and    human neuraminidase 4.-   64. The method according to any one of Clauses 50 to 63, wherein    editing of the ligand by the target cell surface-editing enzyme    enhances natural killer (NK) cell activation by increasing    natural-killer group 2 member D protein (NKG2D) binding to a NKG2D    ligand on the target cell surface.-   65. The method according to any one of Clauses 45 to 64, wherein the    individual has cancer, and wherein the conjugate comprises a    targeting moiety that binds to a tumor-associated cell surface    molecule or tumor-specific cell surface molecule on the surface of a    cancer cell of the individual.-   66. The method according to Clause 65, wherein the cancer cell is a    carcinoma cell.-   67. The method according to Clause 65 or Clause 66, wherein the    cancer cell is selected from the group consisting of: a breast    cancer cell, an ovarian cancer cell, a gastric cancer cell, and a    colon cancer cell.-   68. The method according to Clause 66 or Clause 67, wherein the cell    surface molecule is human epidermal growth factor receptor 2 (HER2).-   69. The method according to Clause 68, wherein the targeting moiety    is trastuzumab.-   70. The method according to any one of Clauses 45 to 63, wherein the    targeting moiety is selected from the group consisting of:    cetuximab, daratumumab, girentuximab, panitumumab, ofatumumab, and    rituximab.-   71. A kit comprising the conjugate of any one of Clauses 1 to 42 or    a composition of Clause 43 or Clause 44.-   72. The kit of Clause 71, wherein the kit comprises the conjugate or    composition in one or more unit dosages.-   73. The kit of Clause 72, wherein the kit comprises the conjugate or    composition in two or more unit dosages.-   74. The kit of any one of Clauses 71 to 73, comprising instructions    for using the conjugate or composition to treat an individual in    need thereof.-   75. The kit of Clause 74, wherein the individual has cancer, and the    instructions are for administering to the individual a    therapeutically effective amount of the conjugate or composition to    treat the cancer.-   76. A method, comprising: conjugating a target cell surface-editing    enzyme to a targeting moiety that binds to a cell surface molecule    on the surface of a target cell.-   77. The method according to Clause 76, wherein the conjugating    comprises site-specifically conjugating the target cell    surface-editing enzyme to the targeting moiety.-   78. The method according to Clause 77, wherein the conjugating    comprises site- specifically conjugating the target cell    surface-editing enzyme to a non-natural amino acid of the targeting    moiety.-   79. The method according to any one of Clauses 76 to 78, wherein the    target cell surface-editing enzyme is conjugated to the targeting    moiety via a linker.-   80. The method according to Clause 79, wherein the linker comprises    polyethylene glycol (PEG).-   81. The method according to Clause 79, wherein the linker is a    peptide.-   82. The method according to Clause 81, wherein the conjugate is a    fusion protein.-   83. The method according to any one of Clauses 76 to 80, wherein the    target cell surface-editing enzyme is a sialidase and the targeting    moiety is an antibody.-   84. The method according to Clause 83, wherein the antibody is an    anti-HER2 antibody.-   85. The method according to Clause 84, wherein the antibody is    trastuzamab.-   86. The method according to Clause 83, wherein the antibody is    selected from the group consisting of: cetuximab, daratumumab,    girentuximab, panitumumab, ofatumumab, and rituximab.-   87. A nucleic acid encoding the fusion protein of Clause 82.-   88. An expression vector comprising a promoter operably linked to    the nucleic acid of Clause 87.-   89. A host cell comprising the nucleic acid of Clause 87 or the    expression vector of Clause 88.-   90. The host cell of Clause 89, wherein the host cell is a mammalian    host cell.

Accordingly, the preceding merely illustrates the principles of thepresent disclosure. It will be appreciated that those skilled in the artwill be able to devise various arrangements which, although notexplicitly described or shown herein, embody the principles of theinvention and are included within its spirit and scope. Furthermore, allexamples and conditional language recited herein are principallyintended to aid the reader in understanding the principles of theinvention and the concepts contributed by the inventors to furtheringthe art, and are to be construed as being without limitation to suchspecifically recited examples and conditions. All statements hereinreciting principles, aspects, and embodiments of the invention as wellas specific examples thereof, are intended to encompass both structuraland functional equivalents thereof. It is intended that such equivalentsinclude currently known equivalents and equivalents developed in thefuture, i.e., any elements developed that perform the same function,regardless of structure. The scope of the present invention, therefore,is not intended to be limited to the exemplary embodiments shown anddescribed herein. Rather, the scope and spirit of present invention isembodied by the appended claims.

What is claimed is:
 1. A conjugate, comprising: a targeting moiety thatbinds to a cell surface molecule of a target cell; and a target cellsurface-editing enzyme.
 2. The conjugate of claim 1, wherein thetargeting moiety is selected from the group consisting of: an antibody,a ligand, an aptamer, a nanoparticle, and a small molecule.
 3. Theconjugate of claim 2, wherein the targeting moiety is an antibody. 4.The conjugate of claim 3, wherein the antibody is an IgG, a single chainFv (scFv), Fab, (Fab)₂, or (scFv′)₂.
 5. The conjugate of claim 3,wherein the antibody is an IgG1.
 6. The conjugate of any one of claims 3to 5, wherein the antibody is a monoclonal antibody.
 7. The conjugate ofany one of claims 3 to 6, wherein the antibody is a humanized or humanantibody.
 8. The conjugate of any one of claims 3 to 7, wherein thetarget cell surface-editing enzyme is conjugated to a light chain of theantibody.
 9. The conjugate of any one of claims 3 to 7, wherein thetarget cell surface-editing enzyme is conjugated to a heavy chain of theantibody.
 10. The conjugate of claim 9, wherein the target cellsurface-editing enzyme is conjugated to an Fc region of the antibody.11. The conjugate of claim 9, wherein the target cell surface-editingenzyme is conjugated to the C-terminus of the heavy chain.
 12. Theconjugate of any one of claims 1 to 11, wherein the target cellsurface-editing enzyme is site-specifically conjugated to the targetingmoiety.
 13. The conjugate of claim 12, wherein the targeting moietycomprises a non-natural amino acid to which the target cellsurface-editing enzyme is site-specifically conjugated.
 14. Theconjugate of any one of claims 1 to 13, wherein the target cellsurface-editing enzyme is conjugated to the targeting moiety via alinker.
 15. The conjugate of claim 14, wherein the linker comprisespolyethylene glycol (PEG).
 16. The conjugate of claim 14, wherein thelinker is a peptide.
 17. The conjugate of claim 16, wherein theconjugate is a fusion protein.
 18. The conjugate of any one of claims 1to 15, wherein the target cell is selected from the group consisting of:a cancer cell, an immune cell, and an endothelial cell.
 19. Theconjugate of claim 18, wherein the target cell is a cancer cell.
 20. Theconjugate of claim 19, wherein the cell surface molecule is atumor-associated cell surface molecule.
 21. The conjugate of claim 19,wherein the cell surface molecule is a tumor-specific cell surfacemolecule.
 22. The conjugate of any one of claims 19 to 21, wherein thecancer cell is a carcinoma cell.
 23. The conjugate of any one of claims19 to 22, wherein the cancer cell is selected from the group consistingof: a breast cancer cell, an ovarian cancer cell, a gastric cancer cell,and a colon cancer cell.
 24. The conjugate of claim 22 or claim 23,wherein the cell surface molecule is human epidermal growth factorreceptor 2 (HER2).
 25. The conjugate of claim 24, wherein the targetingmoiety is trastuzumab.
 26. The conjugate of any one of claims 3 to 18,wherein the targeting moiety is selected from the group consisting of:cetuximab, daratumumab, girentuximab, panitumumab, ofatumumab, andrituximab.
 27. The conjugate of any one of claims 1 to 26, wherein thetarget cell surface-editing enzyme cleaves a molecule on the surface ofthe target cell, oxidizes a molecule on the surface of the target cell,reduces a molecule on the surface of the target cell, adds a moiety to amolecule on the surface of the target cell, or removes a moiety from amolecule on the surface of the target cell.
 28. The conjugate of any oneof claims 1 to 26, wherein the target cell surface-editing enzymecleaves a molecule on the surface of the target cell.
 29. The conjugateof claim 28, wherein the molecule on the surface of the target cell is aligand.
 30. The conjugate of claim 29, wherein the ligand is a ligand ofan inhibitory immune receptor.
 31. The conjugate of claim 30, whereinthe inhibitory immune receptor is present on an immune cell selectedfrom the group consisting of: a natural killer (NK) cell, a macrophage,a monocyte, a neutrophil, a dendritic cell, a T cell, a B cell, a mastcell, a basophil, and an eosinophil.
 32. The conjugate of claim 31,wherein the inhibitory immune receptor is a sialic acid-binding Ig-likelectin (Siglec) receptor.
 33. The conjugate of claim 32, wherein theSiglec receptor is Siglec
 7. 34. The conjugate of claim 32, wherein theSiglec receptor is Siglec
 9. 35. The conjugate of any one of claims 29to 34, wherein the ligand is a sialoglycan.
 36. The conjugate of any oneof claims 1 to 35, wherein the target cell surface-editing enzyme is asialidase.
 37. The conjugate of claim 36, wherein the sialidase is aSalmonella typhimurium sialidase.
 38. The conjugate of claim 36, whereinthe sialidase is a Vibrio cholerae sialidase.
 39. The conjugate of claim36, wherein the sialidase is a mammalian neuraminidase.
 40. Theconjugate of claim 39, wherein the mammalian neuraminidase is a humanneuraminidase.
 41. The conjugate of claim 40, wherein the humanneuraminidase is selected from the group consisting of: humanneuraminidase 1, human neuraminidase 2, human neuraminidase 3, and humanneuraminidase
 4. 42. The conjugate of any one of claims 1 to 41,comprising two or more target cell surface-editing enzymes conjugated tothe targeting moiety.
 43. A composition, comprising: a conjugate of anyone of claims 1 to 42; and a pharmaceutically acceptable carrier. 44.The composition of claim 43, wherein the composition is formulated forparenteral administration.
 45. A method comprising administering to anindividual in need thereof a conjugate of any one of claims 1 to 42 or acomposition of claim 43 or claim
 44. 46. A method of treating cancercomprising administering to an individual having cancer a conjugate ofany one of claims 1 to 42 or a composition of claim 43 or claim
 44. 47.A method of enhancing antibody-dependent cellular cytotoxicity (ADCC)comprising administering to an individual in need of ADCC a conjugate ofany one of claims 1 to 39 or a composition of claim 40 or claim
 41. 48.The method according to any one of claims 45 to 47, wherein theadministering modulates an immune pathway in the individual.
 49. Themethod according to claim 48, wherein the immune pathway is selectedfrom the group consisting of: an inhibitory immune receptor pathway, acomplement pathway, a paired immunoglobulin-like type 2 receptor (PILR)pathway, and a natural-killer group 2 member D protein (NKG2D) pathway.50. The method according to any one of claims 45 to 49, wherein thetarget cell comprises a ligand on its surface, and the administeringresults in editing of the ligand by the target cell surface-editingenzyme.
 51. The method according to claim 50, wherein the editing of theligand comprises cleavage of all or a portion of the ligand.
 52. Themethod according to claim 50 or claim 51, wherein the ligand is a ligandof an inhibitory immune receptor.
 53. The method according to claim 52,wherein the inhibitory immune receptor is present on an immune cellselected from the group consisting of: a natural killer (NK) cell, amacrophage, a monocyte, a neutrophil, a dendritic cell, a T cell, a Bcell, a mast cell, a basophil, and an eosinophil.
 54. The methodaccording to claim 53, wherein the inhibitory immune receptor is asialic acid-binding Ig-like lectin (Siglec) receptor.
 55. The methodaccording to claim 54, wherein the Siglec receptor is Siglec
 7. 56. Themethod according to claim 54, wherein the Siglec receptor is Siglec 9.57. The method according to any one of claims 50 to 56, wherein theligand is a sialoglycan.
 58. The method according to claim 57, whereinthe target cell surface-editing enzyme is a sialidase.
 59. The methodaccording to claim 58, wherein the sialidase is a Salmonella typhimuriumsialidase.
 60. The method according to claim 58, wherein the sialidaseis a Vibrio cholerae sialidase.
 61. The method according to claim 58,wherein the sialidase is a mammalian neuraminidase.
 62. The methodaccording to claim 61, wherein the mammalian neuraminidase is a humanneuraminidase.
 63. The method according to claim 62, wherein the humanneuraminidase is selected from the group consisting of: humanneuraminidase 1, human neuraminidase 2, human neuraminidase 3, and humanneuraminidase
 4. 64. The method according to any one of claims 50 to 63,wherein editing of the ligand by the target cell surface-editing enzymeenhances natural killer (NK) cell activation by increasingnatural-killer group 2 member D protein (NKG2D) binding to a NKG2Dligand on the target cell surface.
 65. The method according to any oneof claims 45 to 64, wherein the individual has cancer, and wherein theconjugate comprises a targeting moiety that binds to a tumor-associatedcell surface molecule or tumor-specific cell surface molecule on thesurface of a cancer cell of the individual.
 66. The method according toclaim 65, wherein the cancer cell is a carcinoma cell.
 67. The methodaccording to claim 65 or claim 66, wherein the cancer cell is selectedfrom the group consisting of: a breast cancer cell, an ovarian cancercell, a gastric cancer cell, and a colon cancer cell.
 68. The methodaccording to claim 66 or 67, wherein the cell surface molecule is humanepidermal growth factor receptor 2 (HER2).
 69. The method according toclaim 68, wherein the targeting moiety is trastuzumab.
 70. The methodaccording to any one of claims 45 to 63, wherein the targeting moiety isselected from the group consisting of: cetuximab, daratumumab,girentuximab, panitumumab, ofatumumab, and rituximab.
 71. A kitcomprising the conjugate of any one of claims 1 to 42 or a compositionof claim 43 or claim
 44. 72. The kit of claim 71, wherein the kitcomprises the conjugate or composition in one or more unit dosages. 73.The kit of claim 72, wherein the kit comprises the conjugate orcomposition in two or more unit dosages.
 74. The kit of any one ofclaims 71 to 73, comprising instructions for using the conjugate orcomposition to treat an individual in need thereof.
 75. The kit of claim74, wherein the individual has cancer, and the instructions are foradministering to the individual a therapeutically effective amount ofthe conjugate or composition to treat the cancer.
 76. A method,comprising: conjugating a target cell surface-editing enzyme to atargeting moiety that binds to a cell surface molecule on the surface ofa target cell.
 77. The method according to claim 76, wherein theconjugating comprises site-specifically conjugating the target cellsurface-editing enzyme to the targeting moiety.
 78. The method accordingto claim 77, wherein the conjugating comprises site-specificallyconjugating the target cell surface-editing enzyme to a non-naturalamino acid of the targeting moiety.
 79. The method according to any oneof claims 76 to 78, wherein the target cell surface-editing enzyme isconjugated to the targeting moiety via a linker.
 80. The methodaccording to claim 79, wherein the linker comprises polyethylene glycol(PEG).
 81. The method according to claim 79, wherein the linker is apeptide.
 82. The method according to claim 81, wherein the conjugate isa fusion protein.
 83. The method according to any one of claims 76 to80, wherein the target cell surface-editing enzyme is a sialidase andthe targeting moiety is an antibody.
 84. The method according to claim83, wherein the antibody is an anti-HER2 antibody.
 85. The methodaccording to claim 84, wherein the antibody is trastuzamab.
 86. Themethod according to claim 83, wherein the antibody is selected from thegroup consisting of: cetuximab, daratumumab, girentuximab, panitumumab,ofatumumab, and rituximab.
 87. A nucleic acid encoding the fusionprotein of claim
 82. 88. An expression vector comprising a promoteroperably linked to the nucleic acid of claim
 87. 89. A host cellcomprising the nucleic acid of claim 87 or the expression vector ofclaim
 88. 90. The host cell of claim 89, wherein the host cell is amammalian host cell.